\.  S 


PUBLIC  HEALTH 

CHEMISTRY  AND  BACTERIOLOGY 

A     HANDBOOK 
FOR     D.P.H.     STUDENTS 


DAVID    McKAIL, 

M.D.(GLASG.),    D.P.H. (CAMB.),    F.R.F.P.S.G. 

Lecturer  on  Public  Health  and  Forensic  Medicine,  St.  Mungo's  College, 

Glasgow ;     Lecturer  on   Hygiene  to  Nurses,  Glasgow   Royal 

Infirmary ;     Assistant    Lecturer    on    School   Hygiene  to 

Teachers  in  Training,  Glasgow  Provincial  Committee  ; 

Examiner  in  Public  Health  for  the   D.P.H., 

Scottish  Conjoint  Board ;  Part-time  School 

Doctor,  Glasgozv   School  Board 


mew   Jtjorfe: 
WILLIAM     WOOD     AND     COMPANY 

MDCCCCXII 


LIDflAlU'  - 
PUBLIC 
HiALTH 
LIBRARY 


JOHN    WRIGHT    AND    SONS    LTD. 
PRINTERS,     BRISTOL. 


PREFACE 

The  present  work  is  based  on  the  notes  prepared  by 
the  writer  for  his  class  while  teaching  Public  Health 
Chemistry  and  Bacteriology.  These  were  compiled  from 
various  standard  works,  and  from  articles  appearing  in 
the  medical  journals  from  time  to  time.  No  originality 
is  therefore  claimed  for  them,  and  grateful  acknowledg- 
ment is  made  to  the  sources  indicated.* 

The  book  is  intended  to  assist  in,  and  supplement, 
actual  laboratory  teaching,  and  not  in  any  way  to  super- 
sede it.  It  has  been  the  writer's  endeavour  therefore  to 
make  it  as  complete  as  possible,  while  leaving  out 
matters  which  can  be  more  satisfactorily  taught  and 
demonstrated  than  written  about.  No  illustrations  have 
been  introduced,  as  the  various  instruments  and  apparatus 
are  seen  and  used  in  the  actual  work. 

I  desire  to  express  my  hearty  appreciation  of,  and  my 
best  thanks  for,   the   assistance  received    from  a    number 


*  I  am  especially  indebted  to  the  following  works  : — Pakes'  "  Hygiene," 
Notter  &  Firth's  "  Hygiene,"  Moor  &  Partridge's  "  Aids  to  the  Analysis 
of  Foods  and  Drugs,"  Richter's  "  Organic  Chemistry,"  the  "  Harmsworth 
Self -Educator,"  the  "  Encyclopaedia  and  Dictionary  of  Medicine  and 
Surgery,"  Jordan's  "Bacteriology,"  Muir  &  Ritchie's  "  Bacteriology,"  Hiss 
and  Zinsser's  "  Bacteriology,"  Abel's  "  Laboratory  Handbook,"  Stitt's 
"  Practical  Bacteriology," SimsWoodhead's  "Bacteria and  their  Products," 
and  the  Lancet,  Public  Health,  and  the  Bacteriological  Supplement  to  the 
Medical  Officer. 

259680 


iv  PREFACE 

of  friends,  and  very  specially  from  Dr.  R.  M.  Buchanan, 
Bacteriologist  to  the  Corporation  of  Glasgow,  and  Dr.  J. 
Hume  Patterson,  Bacteriologist  to  the  County  Council  of 
Lanarkshire.  To  Dr.  John  A.  Wilson,  Assistant  Bacteri- 
ologist, Glasgow,  I  owe  a  careful  revision  of  the  chapter 
on  Special  Bacteriological  Examinations,  including  the 
Table  on  pp.  354—5,  and  much  helpful  criticism.  To  Dr. 
Archibald  MacMillan  I  am  indebted  for  criticism  of  some 
parts  of  the  Chemical  portion  of  the  book,  as  a  result  of 
which  certain  changes  have  been  made. 

DAVID  McKAIL. 
Glasgow,  1912. 


CONTENTS 

PAGES 

Preface  ------        iii-iv 

Introduction  _.-...  1-4 

Part  I.— PUBLIC    HEALTH    CHEMISTRY. 

Chap.  I. — Chemical  Analysis  -  -  -         5-18 

Standard  solutions,  6  ;  Normal  solutions,  7  ;  Indicators, 
10  ;  Acidimetry  and  alkalinity,  12  ;  Weighing  and 
measuring,  14. 

Chap.  II. — Water  Analysis  -  -  -       19-61 

Introductory,  19  ;  Collection  of  sample,  21  ;  Physical 
examination,  22  ;  Chemical  examination,  22  ;]  Quanti- 
tative estimation  of  lime,  magnesia,  phosphates  and 
sulphates,  30  ;  Hardness,  34  ;  Organic  matter,  38  ; 
Nitrites  and  nitrates,  47  ;  Ice,  53  ;  Mineral  waters  and 
aerated  waters,  53  ;  Interpretation  of  results,  53  ; 
Sewage  and  sewage  effluents,  56. 

Chap.  III. — Examination  of  Air     -  62-70 

Collection,  62  ;  Odour,  temperature,  pressure  and 
humidity,  63  ;  Carbonic  acid  gas,  63  ;  Ozone,  66  ;  Oxid- 
izable  and  organic  matter,  66  ;  Noxious  emanations,  67  ; 
Suspended  matter,  69  ;  Carbon  monoxide,  69  ;  Oxygen, 
69  ;    Gases  in  mines,  70. 

Chap.  IV.— Soils        -  -  -.  -  -      71,  72 

Examination  of  sample  :  Ground  air,  71  ;  Ground  water, 
72  ;    Soil  temperature,  72. 

Chap.  V.— Foods        -  -  -  -  -     73-112 

Milk,  73  ;  Cream,  condensed  milk,  infant  foods,  84  ; 
Butter,  87  ;  Cheese,  95  ;  Cereals,  96  ;  Starches,  101  ; 
Carbohydrates,  102  ;  Golden  syrup,  honey,  no  ;  Mustard, 
pepper,  ginger,  peas,  meat  extracts,  112. 

Chap.  VI. — Beverages  -  1 13-134 

Coffee,  113;  Tea,  115;  Cocoa,  117;  Lemon  juice  and 
lime  juice,  119  ;  Vinegar,  122  ;  Beer,  125  ;  Wine,  130  ; 
Spirits,  132. 


vi  CONTENTS 

PAGES 

Chap.  VII. — Disinfectants,     Antiseptics,     and 

Deodorants  -  135-143 

Bleaching  powder,  135  ;  Formalin,  136  ;  Permanganate 
of  potash,  136  ;  Ferrous  sulphate,  137  ;  Carbolic  acid, 
137  ;  Carbolic  powders,  138  ;  Sodium  sulphite  and 
sulphurous  acid,  139  ;  Zinc  chloride,  139  ;  Copper 
sulphate,  139  ;   The  Lancet-acetone-baryta  method,  140. 

Chap.  VIII. — Appendix  -  144-147 

Table  of  Glaisher's  factors,  144  ;  Weight  of  cubic  foot  of 
water  vapour  at  various  temperatures,  145  ;  Alcohol 
tables,  145-147. 


Part  II.— PUBLIC    HEALTH    BACTERIOLOGY. 
Introductory  -  148-151 

Chap.  IX. — General  Principles      -  -  -   151-171 

Bacteriological  media,  151  ;  Sterilization  and  disinfec- 
tion, 155  ;  Cultural  methods,  155  ;  Modes  of  study,  156  ; 
Cultural  reactions,  156  ;  Liquefaction  of  gelatin,  159  ; 
Haemolysis,  160  ;  Staining  reactions  and  methods,  161  ; 
Polychrome  stains,  167;  Inoculation  of  animals,  169; 
Unicellular  micro-organisms,  170. 

Chap.  X. — Results  of  Bacterial  Activity  -   172-179 

Products,  172  ;    Infection,  173  ;    Bacterial  poisons,  175. 

Chap.  XL — Immunity  and  Anaphylaxis     -  -  180-219 

Immunity,  181  ;  Immunity  phenomena,  191  5  Theories 
of  immunity,  198  ;  Further  immunity  phenomena,  204  ; 
Anaphylaxis,    212. 

Chap.  XII. — Micrococci        -  220-229 

Staphylococci,  220  ;  Streptococci,  222  ;  Pneumococcus, 
224  ;  Streptococcus  mucosus,  226  ;  Meningococcus,  226  ; 
Gonococcus,  227  ;  Micrococcus  tetragenus,  Micrococcus 
catarrhalis,   Micrococcus  melitensis,  228. 

Chap.  XIII. — Non-sporing  Bacilli  -  -  230-303 

The  Colon-typhoid-dysentery  group,  230 ;  Capsulated 
bacilli,  240 ;  Bacillus  acidi  lactici  (Hueppe),  242  ; 
Minute  bacilli,  243  ;  Morax-Axenfeld  diplo-bacillus, 
245  ;  Diphtheria  bacillus,  246  ;  Bacillus  mallei,  250  ; 
Bacillus  pestis,  254  ;  Summary  of  Lancet  reports  of 
plague  in  China,  1910-11,  263  ;  Tubercle  bacillus,  273  ; 
Other  acid-fast  bacilli,  284  ;  Actinomycosis,  or  Ray 
fungus  disease,  286  ;  Summary  of  final  report  of  British 
Royal  Commission  on  Tuberculosis,  288. 


CONTENTS  vii 

PAGES 

Chap.  XIV. — Sporing  Bacilli  -  -  -  304-320 

Spore-bearing  aerobic  bacilli,  304  ;  Spore-bearing 
anaerobic  bacilli,  310 

Chap.  XV. — Spirilla  -  321-325 

Spirillum  choleras  Asiaticae,  321  ;  Spirilla  other  than  the 
cholera  spirillum,  324. 

Chap.  XVI. — Spirochetes    -  326-329 

Spirochaeta  recurrentis,  326  ;  Spirochagta  Vincenti,  327  ; 
The  micro-organism  of  Syphilis,  327  ;    Yaws,  329. 

Chap.  XVII. — Yeasts  and  Moulds  -  -  330-344 

Yeasts,  331  ;    Moulds,  334. 

Chap.  XVIII. — Special  Bacteriological  Examina- 
tions -  345-383 

Water,  345  ;  Air,  363  ;  Soil,  365  ;  Dust,  sewage  and 
sewage  effluents,  and  excremental  matters,  365  ;  Milk, 
366 ;  Butter,  Cheese,  Shellfish,  Watercress  and  other 
vegetables,  376  ;    Disinfectants,  377. 

Appendix         __..--  384-392 

Regulations  for  the  Diploma  in  Public  Health,  384 ; 
Preservatives  in  milk  and  cream,  388  ;  Bovine  and  human 
types  of  Tubercle  bacilli,  390. 

Index  -  •  393 


Addenda    and    Erratum 

Page  38,  line  7  : 

after  ' '  present  " 

add    "  as  chloride,  sulphate,  etc." 

Page    79,    Preservatives  in   Milk  :     See  new  Regulations  (1912)  on 
page  388. 

Page  80,  line  30  : 

after  ' '  tubes  " 

add    "  plus  a  drop  of  phenolphthalein  solution  " 

Page  84,  Preservatives  in  Cream  :    See  new  Regulations  (1912)  on 
page  388. 

Page  233,    Antityphoid  vaccine  :    Leishman   sterilizes   the   typhoid 
culture  at  53°  C  for  one  hour* 

Page  277,  line  2  from  foot : 
after  "  growth  " 
add    "  on  glycerin  egg  medium  (see  page  391)  " 

Page  257,  line  5  from  foot  : 

for  "  Limond,"  read  "  Simond  " 


PUBLIC  HEALTH 
CHEMISTRY  AND  BACTERIOLOGY. 


INTRODUCTORY. 

pUBLIC  HEALTH  Chemistry  and  Bacteriology  do  not 
r-*-  differ  fundamentally  from  general  chemistry  and 
bacteriology,  and  in  fact  are  based  on  these  subjects,  of 
which  they  are  specialized  departments.  The  same 
principles  underlie  the  part  as  the  whole,  and  the  accumu- 
lation of  scraps  of  knowledge  derived  from  the  parent 
sciences,  under  the  heading  of  Public  Health  Chemistry 
and  Bacteriology,  is  justifiable  only  on  the  score  of  con- 
venience and  the  importance  of  economizing  the  student's 
time.  It  is  therefore  necessary  to  remember  that  the 
whole  is  greater  than  the  part,  and  that  to  have  wide  and 
luminous  views  of  the  subjects  so  designated,  the  study 
of  them  should  not  be  strictly  utilitarian,  but  be  extended 
in  all  necessary  directions  as  much  as  possible. 

A  knowledge  of  Public  Health  Chemistry  and  Bacteri- 
ology has  become  more  urgently  called  for,  owing  to  the 
increase  of  Public  Health  work,  to  participate  in  which 
it  is  necessary  to  possess  a  Diploma  in  Public  Health. 

The  General  Medical  Council  at  their  meeting  on  ist 
December,  191 1,  adopted  in  an  amended  form  the  resolu- 
tions and  rules  which  form  the  Regulations  for  the  Diploma 
in  Public  Health.  These  are  printed  in  full  in  an  Appendix 
to  this  volume.  For  our  present  purpose  it  is  sufficient  to 
quote  here  Rule  2. 

Rule  2.  Every  Candidate  for  a  Diploma  in  Sanitary  Science, 
Public  Health,  or  State  Medicine  shall  have  produced  satisfactory 
evidence  that,  after  obtaining  a  registrable  qualification,  which 
should  be  registered  before  admission  to  examination  for  the 
diploma,  he  has  received  practical  instruction  in  a  laboratory  or 


2  INTRODUCTORY 

laboratories,  British  or  foreign,  approved  by  the  licensing  body 
granting  the  diploma,  [in  which  chemistry,  bacteriology,  and  the 
pathology  of  the  diseases  of  animals  transmissible  to  man  are 
taught. 

Note. — The  laboratory  instruction  shall  cover  a  period  of  not  less 
than  four  calendar  months,  and  the  candidate  shall  produce 
evidence  that  he  has  worked  in  the  laboratory  for  at  least  240  hours, 
of  which  not  more  than  one-half  shall  be  devoted  to  practical 
chemistry.  The  laboratory  course  should  be  so  arranged  as  to  lay 
special  stress  on  work  which  bears  most  directly  on  the  duties  of 
a  medical  officer  of  health. 

The  amendments  in  this  rule  reduce  the  number  of 
months  of  instruction  from  six  to  four,  and  define  the 
hours  worked  as  240,  of  which  at  least  120  shall  be  devoted 
to  Bacteriology.  The  net  result  is  that  students  will 
thus  require  to  do  as  a  minimum  15  hours'  practical 
laboratory  work  per  week  for  four  months.  Many  students 
will  find  it  more  convenient  to  do  12  hours  per  week  for 
a  so-called  six  months'  term  of  20  weeks. 

The  final  clause  of  the  rule,  namely,  "  The  laboratory 
course  should  be  so  arranged  as  to  lay  special  stress 
on  work  which  bears  most  directly  on  the  duties  of 
a  medical  officer  of  health "  might  usefully  have  been 
made  more  specific,  and  been  extended  to  include  the 
phrase,  "  and  the  Examiners  for  the  Diploma  shall  have 
special  regard  to  this  recommendation." 

The  student  will  "be  well  advised  to  exceed  the  minimum 
periods  above  laid  down,  as  far  as  he  can,  and  more  espe- 
cially if  he  is  not  already  well  informed  in  the  subjects  of 
chemistry  and  bacteriology. 

The  scope  of  the  work  and  the  methods  may  be  thus 
summarized  on  the  basis  of  the  Syllabus  of  the  Scottish 
Conjoint  Board : — 

DIPLOMA    IN    PUBLIC   HEALTH. 
Public  Health  Laboratory  Work  Course 
comprises  : — 

Physical,  Chemical,  and  Bacteriological  Examination  of 
water,  sewage  and  sewage  effluents,  air  and  other  gases, 
food  stuffs,  beverages,  soils,  and  building  materials. 
Examination  of  disinfectants,  antiseptics,  deodorants. 


INTRODUCTORY  3 

Detection  of  Poisons  in  foods,  dress,  decoration. 

Examination  of  parasites  and  other  animal  organisms 
found  in  the  body  and  human  food  stuffs. 

Bacteriology  and  Bacteriological  Methods :  apparatus, 
media,  modes  of  culture  ;  culture  and  recognition  of  the 
principal  pathogenic  organisms  ;  bacteriological  examina- 
tion of  water,  air,  and  foods ;  antitoxins ;  principles 
of  serumtherapy  and  immunization ;  cultivation  and 
recognition  of  micro-organisms  in  relation  to  epidemic  and 
other  diseases. 

Modes  of  Examination. 

Physical. — Inspection  by  the  unaided  senses — colour, 
odour,  taste,  transparency,  turbidity,  etc.  ;  specific 
gravity  ;  microscopic  examination  ;  spectroscopic  examin- 
ation ;    polariscopic  examination. 

Chemical. — Examination  for  proximate  principles,  e.g., 
water,  solids,  fat,  sugar,  etc.  ;  search  for  and  identification 
of  chemical  impurities  such  as  ammonia,  lead,  borax,  etc.  ; 
quantitative  estimations  of  above. 

Bacteriological. — Enumeration  of  bacteria  present  ; 
search  for  pathogenic  forms ;  if  found,  isolation  and 
identification. 


Part  I. 
PUBLIC     HEALTH     CHEMISTRY. 

IT  is  necessary,  in  the  first  place,  to  consider  carefully 
certain  points  of  chemical  practice,  which  form  the 
basis  of  much  of  the  subsequent  work,  and  which  must 
be  thoroughly  understood  to  enable  the  latter  to  be 
readily  followed  and  apprehended. 


CHAPTER     I, 
CHEMICAL   ANALYSIS. 

This  is  of  two  kinds — Qualitative  or  Quantitative. 

1.  Qualitative. — Consists  in  proving  the  presence  or 
absence  of  certain  metals  or  salts,  or  generally  of  chemical 
elements  or  radicles  or  compounds  in  a  substance  or 
solution,  by  the  use  of  a  series  of  tests. 

2.  Quantitative. — Consists  in  separating  out  the  con- 
stituents of  any  composite  body  and  accurately  estimating 
the  amount  of  each  of  them.  This  may  be  done  in  three 
ways  :    Gravi  metrically,  Volumetrically,  Colorimetrically. 

Gravimetric  method. — The  desired  constituent  is  separated 
out  in  a  known  form,  and  this  is  accurately  weighed.  As  a 
method  it  is  often  very  complicated,  very  lengthy,  and 
requires  elaborate  apparatus  and  much  skill. 

Volumetric  method. — This  consists  in  submitting  the 
substance  to  certain  characteristic  reactions,  a  measured 
quantity  of  a  solution  of  known  strength  being  added 
until  a  change  looked  for  occurs.  From  the  quantity  of 
reagent  used,  the  amount  of  the  substance  found  can  be 
calculated  by  known  chemical  laws.  It  is  less  elaborate, 
much  more  quickly  accomplished,  needs  simpler  apparatus 
as  a  rule,  is  susceptible  of  great  accuracy,  and  the  skill 
required  is  less  specialized. 

Colorimetric  method. — This  consists  in  using  a  reaction 
which  produces  a  coloured  tint,  which  is  compared  with 
the  tint  obtained  from  the  same  treatment  of  a  known 


G  PUBLIC    HEALTH    CHEMISTRY 

quantity  of  the  substance  under  investigation.  Exact 
matching  of  the  tints  is  arrived  at,  either  by  dilution  of 
the  stronger,  or  by  putting  up  several  standard  tints. 
Requirements  for  Volumetric  Analysis. — 
i.  Solution  of  reagent  or  test,  the  chemical  power  of 
which  is  accurately  known  ;  this  is  called  the  Standard 
Solution. 

2.  A  graduated  vessel,  from  which  portions  of  the 
standard  solution  may  be  accurately  delivered  :  the  Burette. 

3.  Some  indication,  unmistakeable  to  the  eye,  that  the 
reaction  is  terminated  or  concluded  :    the  Indicator. 

The  process  is  called,  a  titration,  i.e.,  an  estimation  of 
the  titre  or  strength  of  a  solution,  and  the  person  is  said 
to  titrate  the  solution. 

Volumetric  methods  may  be  classified  thus  : — 

1.  Neutralization  of  acids  by  alkalies,  and  vice  versa — 
acidimetry  and  alkalimetry. 

2.  Reduction  or  oxidation  of  substances — for  example, 
ferrous  sulphate  titrated  with  potassium  permanganate 
illustrates  both  actions. 

3.  Precipitation  of  some  insoluble  and  definite  com- 
bination— for  example,  precipitation  of  AgCl  in  testing 
for  chlorides. 

STANDARD     SOLUTIONS. 

A  standard  solution  of  any  substance  is  made  when  a 
known  quantity  of  that  substance  is  dissolved  in  a  known 
quantity  of  water.  Then  the  strength  can  be  expressed 
definitely  as — 

So  many  pounds  per  gallon,  or  pint,  or   ounce  ;   or 
grains  „  „  „        or 

„  grammes  per  litre  or  cubic  centimetre  (c.c.) ;  or 

„  milligrammes      ,,  „  ,, 

Thus  we  might  have  a  standard  solution  of  NaCl  of  these 
strengths  :    (1  gallon  =  10  lbs.  =  70,000  grs.). 

1  lb.  per  gallon  ;    then  1  fluid  grain  =  o-i  grain  NaCl. 
1  drachm  per  fluid  ounce  ;  then  1  minim  =  0-125  gr-  NaCl. 
1   gramme   per  litre ;    then   1   cubic   centimetre  —  o-ooi 
gramme,  or  1  milligramme  of  NaCl  per  c.c. 


CHEMICAL    ANALYSIS  7 

The  metric  system  of  weights  and  measures  is  found  to 
be  very  convenient  for  such  solutions,  because — 
i  gramme  dissolved  in  i  litre  =  i  milligramme  in  i  c.c.  ;  or 
n  grammes  dissolved  in  i  litre  =  n  milligrammes  in  i  c.c. 

Standard  solutions  often  have  their  strength  expressed 
in  terms  of  some  other  substance  which  they  measure — 
usually  an  elementary  substance.     For  example — 

AgN08      +      NaCl      =      AgCl      +      NaN03 
170  (23  +  35-5). 

From  this  equation  we  see  that  170  parts  of  silver 
nitrate  precipitate  completely  58-5  parts  of  sodium 
chloride  containing  35-5  parts  of  chlorine.  If  we  wish 
to  estimate  the  amount  of  CI  present  in  a  solution  of 
unknown  strength,  we  can  titrate  with  a  solution  of  silver 
nitrate  of  known  strength — that  is,  a  standard  solution. 
What  strength  shall  we  make  it  ? 

a.  170  grm.  AgN03  in  1  litre  of  water  will  precipitate 
35-5  grm.  CI ;  then  1  c.c.  will  precipitate  35-5  mgr.  CI. 

b.  17-0  grm.  AgN03  in  1  litre  of  water  will  precipitate 
3-55  grm.  CI ;  then  1  c.c,  will  precipitate  3"55  mgr.  CI. 

c.  J—  =  4-78  grm.   in   1   litre  of  water  will  precipitate 

1  grm.  CI;  then  1  c.c.  will  precipitate  1  mgr.  CI. 

The  strength  chosen  depends  on — (1)  The  simplicity 
desired ;  (2)  The  strength  of  solution  to  be  tested ; 
(3)  Whether  any  one  of  these  strengths  will  be  more 
useful  than  the  others  for  other  estimations,  and  so  save 
needless  duplication  of  solutions. 

NORMAL     SOLUTIONS. 

These  are  standard  solutions  made  to  a  certain  strength 
on  the  basis  of  chemical  theory  and  practice.  Thus,  from 
the  equation — 

NaOH  +  HC1  =  NaCl  +  H20 
40  36-5 

we  see  that  40  parts  of  sodium  hydrate  are  exactly  neutral- 
ized by  36-5  parts  of  hydrochloric  acid.     If  therefore  we 


8  PUBLIC    HEALTH    CHEMISTRY 

make  a  standard  solution  of  NaOH  40  grammes  to  1  litre, 
and  one  of  HC1  36-5  grammes  to  1  litre,  then — 

1  c.c.  stand,  sol.  NaOH  =  1  c.c.  stand,  sol.  HC1, 

and  if  we  can  build  other  solutions  on  the  same  plan,  we 
shall  have  a  whole  range  of  solutions  which  are  chemically 
equivalent  c.c.  for  c.c.  This  will  obviate  having  a  strength 
of  acid  for  titrating  soda,  another  strength  for  potash, 
another  for  baryta,  and  so  on,  and  reversely.  This  result 
is  obtained,  or  rather  attained,  by  using  normal  solutions, 
which  are  thus  defined  : — 

A  normal  solution  is  one  which  contains  in  1  litre  of 

distilled  water  at  160  C.  the  hydrogen  equivalent  of  the 

active  reagent  weighed  in  grammes,  hydrogen  being  taken 

as  one  gramme.     Such  a  solution  is  usually  indicated  by 

N 
the  mark  N  or  — .     If  diluted,  the  degree  of  dilution  is 

indicated  by  a  denominator,  thus — 

N  JN  JN_  _N_ 

2  10  100  1000 

which  are  respectively— 
seminormal       decinormal       centinormal      millinormal 

The  hydrogen  equivalent  of  a  substance  is  found  by 
taking  its  molecular  weight,  the  number  of  atoms  usually 
replaced  by  hydrogen,  and  .the  valency  of  these  atoms. 
Divide  the  molecular  weight  by  the  product  of  the  number 
of  atoms  and  their  valency.     Thus — 

NaCl  m.w.  =58-5  no.  of  replaceable  atoms=i  valency=i  ; 
therefore — 

N/i   NaCl=58«5/ix  1=58-5  grm.  to  1  litre  of  water. 
Similarly — 

HC1  m.w.=36-5  n.r.a.=i  v.=i;  hence 
N/i=36-5  grm.  per  litre. 
H2S04    m.w.  =    98  grm.  N/i  =  49  grm.  per  litre. 
HNO3     m.w.  =    63     „     N/i  =  63  '   „ 
NaOH     m.w.  =    40     ,,     N/i  =  40       ,, 
KOH       m.w.  =    56     „     N/i  =  56.      ;, 
Na2C03  m.w.  =  106    ,,     N/i=-53 
K2C03    m.w.  a  138     „     N/i  =  69 


CHEMICAL    ANALYSIS  9 

Where  the  substance  possesses  molecules  of  water  of 
crystallization,  the  weight  of  these  must  be  added  to  the 
sum  of  the  molecule  proper,  in  order  to  arrive  at  the  right 
figure  for  the  normal  solution.     Thus — 

H2C204+2H20  m.w.  =  i26     N/i=63  grm.  per  litre 
(oxalic  acid). 

H3C6H507+H20  m.w.=2io      N/i=70  grm.  per  litre 
(citric  acid). 

H2C4H406  m.w.  =  i5o     N/i= 75  grm.  per  litre 
(tartaric  acid). 

HC  2H  30  2  m.w.  =60       N  /i  =60  grm.  per  litre 
(acetic  acid). 

Some  salts  with  enlarged  molecules  : — 

Ferrous  sulphate,  FeS04+7  H20  molecular  weight  =278 
Copper  sulphate,  CuSOd-|- 5  H20  „  „      =249 

Lead  acetate,  Pb(C  2H  30  2)  2+3  H  20        ,,  „      =379 

Sodium  thiosulphate,  Na2S20  3+5. H20  ,,  ,,      =248 

The  hydrogen  equivalent  of  some  reagents  is  not  so 
easily  arrived  at ;  for  instance,  potassium  permanganate, 
variously   written   as 

K2Mn208     and     KMn04. 

The  molecular  weight  of  the  first  formula  is  316,  and  of  the 
second  is  158.  Nevertheless,  the  normal  solution  is  not 
158  grammes  per  litre,  but  is  31-6  grammes  per  litre. 
This  is  because  potassium  permanganate  does  not  react — 
as  its  formula  might  suggest — as  a  manganate,  but  reacts 
as  a  double  salt  of  potassium  and  manganese,  as  if  it  were 
written  thus — 

K20  +  Mn207, 

and  in  reaction  the  latter  oxide  is  reduced  and  oxj^gen 
liberated  ;    thus — 

K  2Mn  20  8+3  H  2SO  4=K  2SO  4+2  MnSO  4+3  H  20+5  O 
316  80 

We  here  see  that  316  grammes  of  permanganate  of  potash 
liberate  80  grammes  of  oxygen,  which  are  chemically 
equivalent   to    10   grammes    of    hydrogen,    and   so    31-6 


10  PUBLIC    HEALTH    CHEMISTRY 

grammes    of    the   salt    are   equivalent   to    I    gramme   of 
hydrogen.     Hence — 


N/i 

KMn04 

=  31-6  grm.  per  litre. 

N/io 

do. 

=    3-16  do.         do. 

N/ioo 

do. 

^      -316  grm.    do. 

Non- Standardized   Solutions  : — 

Ammonia  free  water. 

Organically  pure  ammonia  free  water. 

Nessler's  solution. 

Methyl-orange  solution  (1  grm.  per  litre  of  water). 

Phenolphthalein  solution  (1  %  in  50  %  alcohol). 

Starch  solution  (5  grm.  per  litre  of  boiling  water). 

Baryta  water  (5  grm.  of  crystallized  barium  hydrate  in 
1  litre  of  freshly  boiled  distilled  water.  Stopper  and  set 
aside  for  three  days,  and  decant  off  clear  liquid.     About 

Metaphenylene  -  diamine  (5  grm.  per  litre  aq.  dest. 
slightly  acidulated  with  a  few  drops  of  sulphuric  acid). 

Naphthylamine  acetate  in  sulphanilic  acetate  : — 

a.  3  to  4  grm.  of  sulphanilic  acid  dissolved  in   1   litre 

of  dilute  acetic  acid. 

b.  frds  grm.  of  naphthylamine  are  boiled  with   150 

c.c.  of  aq.  dest.,  the  colourless  liquid  poured  off 
and  diluted  to  1  litre  with  dilute  acetic  acid. 

c.  Mix  equal  bulks  as  required  for  testing. 

Phenol-sulphonic  acid  (32  c.c.  of  pure  concentrated 
sulphuric  acid  are  added  to  4  c.c.  of  pure  phenol.  Heat 
to  ioo°  C.  for  2  to  3  hours.  Cool,  and  add  no  c.c.  of 
distilled  water). 

INDICATORS. 

These  may  be  classified  under  two  heads  : — 

(1)  Neutrality  indicators,  which  give  a  special  reaction 

with  acid  or  alkaline  liquids,  or  with  both ;    (2)  All  others, 

such  as  starch,  iodine,  chromate  of  potash,  permanganate 

of  potash,  and  soap  lather. 

Neutrality    indicators    are    divided    thus    by    R.    T. 

Thompson : — 


CHEMICAL    ANALYSIS  11 

(i)  Methyl-orange  group :  are  most  susceptible  to 
alkalies ;  methyl-orange,  lacmoid,  cochineal,  congo-red ; 
(2)  Phenolphthalein  group  :  are  most  susceptible  to  acids  ; 
phenolphthalein,  turmeric  ;  (3)  Litmus  group :  are  inter- 
mediate in  susceptibility — litmus,  rosolic  acid,  phenace- 
tolin. 

Sensitiveness. — Phenolphthalein,  lacmoid,  rosolic  acid, 
and  phenacetolin  showed  change  of  colour  with  one-fifth 
quantity  of  acid  or  alkali  required  by  methyl-orange  and 
litmus  ;  that  is  to  say,  if  the  two  latter  required  in  100 
c.c.  of  acid  or  alkali  0-5  c.c.  to  show  change  of  colour,  the 
former  required  only  o-i  c.c. 

Neutral  point  of  one  indicator  does  not  coincide  exactly 
with  that  of  other  indicators.  Thus,  saliva  is  generally 
neutral  to  litmus,  alkaline  to  lacmoid  or  congo-red,  and 
acid  to  turmeric.     Fresh  milk  shows  similar  variations. 

1.  Litmus  solution  is  violet  coloured.  Acids  change  it 
to  red ;  alkalies  to  blue. 

In  cold  solution  it  may  be  used  for  the  titration  of — 

Hydrates  of  soda,  potash,  ammonia,  lime,  baryta,  etc. 
Nitric,  sulphuric,  hydrochloric,  and  oxalic  acids. 
Arsenites  and  silicates  of  soda  and  potash. 

In  boiling  solution — 

Carbonates  and  bicarbonates  of  K,  Na,  Ca,  Mg,  Ba. 
Sulphides  of  sodium  and  potassium. 

2.  Methyl-orange  is  orange-brown  in  colour.  Acids 
change  it  to  pink  ;  alkalies  to  faint  yellow. 

Only  used  in  cold  solution,  and  then  for  titration  of — 
Hydrates,  carbonates,  bicarbonates  of   K,  Na,  NH3, 

Ca,  Mg,  Ba,  etc. 
Sulphides,  arsenites,  silicates,  borates  of  K,  Na,  NH3, 

Ca,  Mg,  Ba,  etc. 
All  the  mineral  acids. 
Sulphites. 
Half  the  base   in   the  alkaline   and   earthy  alkaline 

phosphates  and  arseniates. 
Not  for  organic  acids,  nor  in  presence  of  nitrous  acid 

or  nitrites,  which  decompose  it. 


12  PUBLIC    HEALTH    CHEMISTRY 

3.  Phenolphthalein  is  colourless  in  solution.  Acids  cause 
no  change  in  colour ;  alkalies  change  it  to  purple-red. 

Used  in  the  cold  for  titration  of — 
Alkaline  hydrates,  except  ammonia. 
Mineral  acids. 
Organic    acids    (oxalic,    tartaric,    acetic,    citric,    and 

others). 
Carbonates  to  bicarbonates. 

May  be  used  in  alcoholic  solutions,  and  hence  for 
organic  acids  insoluble  in  water.  Also  for  acids  combined 
with  bases,  like  morphia,  quinine,  brucine,  etc.,  the  organic 
base  having  no  effect  on  it. 

4.  Rosolic  Acid  is  pale  yellow  in  solution.  With  acids, 
unchanged ;  with  alkalies,  violet-red. 

Good  for  mineral  acids  and  oxalic. 
Not  reliable  for  organic  acids. 

5.  Turmeric,  yellow  in  colour.  With  acids,  bright 
yellow ;  with  alkalies,  reddish-brown. 

6  Lacmoid,  blue  and  red  papers  are  best  form  for  use. 
These  are  an  excellent  substitute  for  methyl-orange  when 
latter  cannot  be  used.  It  is  a  derivative  of  resorcin,  and 
is  allied  to  litmus. 

The  other  indicators  will  be  alluded  to  as  their  use  is 
required. 

Rules  as  to  use  of  indicators  commonly  employed  : — 
Methyl-orange  for  mineral  acids. 
Phenolphthalein  for  organic  acids. 
Litmus  for  organic  acids  in  presence  of  free  CO  2  (e.g. 
in  beer). 

ALKALIMETRY  AND  ACIDIMETRY. 

Perform  the  following  exercises  : — 

1.  Titrate  1  c.c.  50  per  cent  NaOH  diluted  with  a  little 
water  (distilled)  with  N/i  H2S04,  using  a  few  drops  of 
litmus  as  indicator. 

Take  the  NaOH  in  a  porcelain  basin— add  the  water  and 
the  litmus  solution.  Then  take  a  burette  and  fill  it  with 
the  normal  acid  solution  ;   be  careful  that  the  nozzle  is 


CHEMICAL    ANALYSIS  13 

full,  and  that  the  lowest  part  of  the  meniscus  is  opposite 
the  zero  mark. 

Now  add  the  acid  I  c.c.  at  a  time,  stirring  after  each 
addition,  with  a  glass  rod.  Continue  the  process  until  the 
colour  of  the  solution  changes  to  red.  The  result  will  be 
accurate  to  I  c.c.  With  practice  the  end  reaction  can  be 
judged  more  accurately  ;  but,  as  already  mentioned,  a 
certain  quantity  of  acid  is  required  for  change  of  colour, 
and  this  is  greater  with  litmus  than  with  some  other 
indicators. 

Say  that  13  c.c.  of  N/i  H2S04  were  required.  How 
much  NaOH  was  present  in  the  1  c.c.  of  sample  ? 

1  c.c.  N/i  H2S04  =1  c.c.  N/i  NaOH 

But  N/i  NaOH  =  40  grm.  per  litre 

Then  1  c.c.  N/i  NaOH  =  0-040  grm. 

Hence  1  c.c.  N/i  H2S04  =  0-040  grm.  NaOH 
Then  13  c.c.    ,,         ,,      =  0-52  grm.  NaOH. 

But  13  c.c.  were  required  to  neutralize  1  c.c.  of  sample. 

Therefore  1  c.c.  of  sample  contains  o#52  grm.  NaOH, 
or  100  c.c.         ,,         will  contain  52   grm.   NaOH. 

The  discrepancy  between  50  grm.  in  100  c.c.  and  52  grm. 
may  be  due  to  : 

(1)  Inaccuracy  in  making  the  soda  solution  originally  ; 

(2)  Inaccuracy  in  measuring  the  1  c.c.  of  soda  solution  ; 

(3)  Inaccuracy   in    the    strength    of    the    normal    acid 

solution  ; 

(4)  Inaccuracy  in  titration. 

2.  Repeat  the  experiment,  using  1  drop  methyl-orange 
as  indicator. 

3.  Repeat  the  experiment,  using  1  drop  phenolphthalein 
as  indicator. 

4.  Titrate  5  c.c.  25  per  cent  H  2SO  4  diluted  with  a  little 
water,  with  N/i  NaOH,  using  a  few  drops  of  litmus  as 
indicator. 

5.  Repeat  the  experiment,  using  1  drop  methyl-orange 
as  indicator. 

6.  Repeat  the  experiment,  using  1  drop  phenolphthalein 
as  indicator. 


14  PUBLIC    HEALTH    CHEMISTRY 

Calculate  out  strength  of  solution  by  method  similar  to 
above. 

Thus,  say  that  25  c.c.  of  N/i  soda  are  required  to 
neutralize  5  c.c.  of  sample,  said  to  be  25  per  cent  sulphuric 
acid. 

Then  1  c.c.      N/i  soda      =  or  is  chemically  equivalent 

to  1  c.c.  N/i  sulphuric  acid. 
But  N/i  H  2SO  4  sat  98/2  or  4.9  grm.  per  litre. 

Hence  1  c.c.  „  „  =  0-049  grm.  H2S04 
Hence  1  c.c.  N/i  NaOH  =  0-049  &rm-  H  2SO  4 
And  25    c.c.       „        „       =  25  X  0-049  =  I,225  grm- 

H2S04. 

But  25  c.c.  N/i  NaOH  were  required  to  neutralize  5  c.c. 
of  sample  ;  therefore  5  c.c.  of  sample  contain  1-225  grm- 
of  sulphuric  acid,  and  100  c.c.  of  sample  contain  24-5  grm. 
of  sulphuiic  acid  ;  that  is,  if  100  c.c.  are  taken  as  100  grm., 
24*5  per  cent.     The  sources  of  error  are  as  before. 

WEIGHING     AND     MEASURING. 

Measuring  of  Solutions. — Small  quantities,  like  1  c.c, 
2  c.c,  etc,  up  to  25  c.c.  or  50  c.c,  are  most  accurately 
measured  by  pipette,  or  on  some  occasions  by  burette.  For 
larger  amounts,  like  100  c.c,  250  c.c,  500  c.c,  and  1000  c.c, 
flasks  with  a  narrow  neck  and  a  mark  thereon  are  the  best. 
When  extreme  accuracy  is  not  essential,  the  ordinary 
graduated  measure  is  quite  efficient.  Be  careful  in  pipet- 
ting certain  liquids  not  to  get  any  drawn  into  the  mouth. 
Never  pipette  strong  sulphuric  acid  or  ammonia  by  mouth 
suction. 

On  Weighing  with  the  Chemical  Balance. — To  weigh  a 
certain  quantity  of  a  substance  the  necessary  weights  are 
placed  in  the  right-hand  pan  of  the  balance.  Some  of  the 
substance  is  then  placed  in  the  left-hand  pan,  or  preferably 
in  a  watch-glass  of  known  weight,  or  balanced  by  a  similar 
one  in  the  other  pan.  The  handle  or  screw  is  now  turned, 
and  the  balance  put  in  action.  If  the  pointer  swings 
more  on  the  side  away  irom  the  weights,  that  is  more  to 
the  left  side,  the  amount  of  substance  is  too  little.  The 
handle  must  now  be  turned  down,  and  the  balance  thus  placed 


CHEMICAL    ANALYSIS 


15 


at rest ,  before  any  further  manipulations  are  tried.  There- 
after more  of  the  substance  is  added,  and  the  same  tech- 
nique carefully  observed. 

To  obtain  the  weight  of  a  substance  or  dish,  or  both,  these 
are  placed  in  the  left-hand  pan,  and  a  trial  weight  is  put 
on  the  right  pan.  If  this  is  too  much,  the  next  lower 
weight  is  tried,  and  so  on,  being  careful  to  observe  the 
technique  outlined  above.  The  weight  is  read  from  the 
vacant  spaces  in  the  case,  and  checked  on  removing  the 
weights.  Always  use  a  watch-glass  in  weighing  a  salt  or 
substance  not  contained  in  a  vessel  of  some  kind. 


Atomic  Weights 

of  the  elements  the 
recently  assigned. 


Aluminium 

Arsenic 

Barium 

Boron 

Bromine 

Calcium 

Carbon 

Chlorine 

Copper 

Hydrogen   .  . 

Iodine 

Iron 

Lead 

Magnesium . 

Manganese.  . 

Mercury 

Nitrogen     . . 

Oxygen 

Phosphorus 

Potassium 

Silver 

Sodium 

Sulphur 

Tin 

Zinc 


— The  following  table  gives  for  some 
older  atomic  weight,  and  that  more 


OLDER 

ATOMIC  WEIGHT. 

NEWER 

27 

26'9 

75 

74*4 

137 

136-4 

11 

10-9 

80 

79-4 

40 

397 

12 

n-9 

35*5 

35*1 

63 

63-1 

1 

— 

127 

126-0 

56 

55-5 

207 

206-4 

24 

24-18 

55 

54'52 

200 

198-5 

14 

i3'9 

16 

i5'9 

31 

30-8 

39 

38-8 

108 

107-1 

23 

22-9 

32 

3i-8 

118 

ii8-i 

..   .  65 

64-9 

16  PUBLIC    HEALTH    CHEMISTRY 

Volume  and  Density  of  Gases.— All  gaseous  molecules, 
at  the  same  temperature  and  pressure,  occupy  the  same 
volume.  This  is  another  way  of  stating  Avogadro's  law, 
that  "  equal  volumes  of  all  gases  (at  a  temperature  suffi- 
ciently remote  from  their  condensation-point — the  so-called 
permanent  gases)  at  the  same  temperature  and  pressure, 
contain  the  same  number  of  molecules." 

From  this  it  follows,  that  if  we  know  the  volume  occupied 
by  one  gaseous  molecule  at  standard  temperature  and 
pressure,  we  thereby  know  the  volume  of  all  gaseous 
molecules  at  the  same  temperature  and  pressure.  The 
volume  occupied  by  the  molecular  weight  of  hydrogen, 
expressed  as  grammes,  that  is  2  grammes  of  hydrogen  gas 
at  0°  C.  and  760  mm.  pressure,  is  22-32  litres.  Hence  the 
statement,  "  the  molecular  weight  of-  any  gas,  expressed 
in  grammes,  measures  22-32  litres,  at  standard  temperature 
and  pressure,  that  is,  0°  C.  and  760  mm.,  or  320  F.  and 
29-9  inches  of  mercury." 

The  Crith. — One  litre  of  hydrogen  gas  at  standard 
temperature  and  pressure  weighs  0-0896  gramme.  This 
weight  has  been  called  a  crith.  It  follows  from  the  above 
statement  of  Avogadro's  law,  that  one  litre  of  oxygen  gas 
will  contain  the  same  number  of  molecules,  each  one 
16  times  the  weight  of  the  hydrogen  molecule  (as  32  :  2), 
and  hence  the  weight  of  one  litre  of  oxygen  gas  will  be 
16  criths,  or  16  x  0-0896  grm.  Similarly,  one  litre  of  carbon 
dioxide  gas  weighs  22  criths  (as  44  :  2).  The  weight,  there- 
fore, of  one  litre  of  an  elementary  gas  (with  exceptions) 
is  equal  to  its  atomic  weight  in  criths  ;  and  of  one  litre  of 
a  compound  gas,  is  equal  to  half  its  molecular  weight  in 
criths. 

Metric  System. 

The  weights  and  measures  of  the  metric  system  are 
those  used  nowadays  in  most  Public  Health  work,  although 
statements  like  "  grains  per  gallon "  still  persist.  The 
chief  units  employed  are  the  following  : — 

Length. — 

1  metre  =  the  length  of  a  rod  of  platinum  at  the  temperature 
of  me] ting  ice.  This  rod  is  kept  at  Paris,  with  official 
copies  in  the  large  towns.     Equals  39-37  inches. 


CHEMICAL     ANALYSIS 


17 


decimetre   =  ^  of  a  metre. 


centimetre 


TTRT 


of  a  metre, 
millimetre  =  Woo  °*  a  metre, 
micron  (/u)  ==  toVtf  °*  a  millimetre, 
kilometre    =  iooo  metres. 


Mass. — 

gramme  =  the  mass  of  i  cubic  centimetre  of  distilled 
water  at  the  temperature  of  its  maximum  density,  40  C. 
or  39*2°  F.,  1  c.c.  at  160  C.  or  6o*8°  F.  =  0*9989  gramme. 
1  gramme  equals  15*432  grains, 
decigramme   =  -fy  of  a  gramme, 
centigramme  =  ^  of  a  gramme, 
milligramme  =  y^Vo-  °f  a  gramme, 
kilogramme    =  1000  grammes. 

Volume. — 

litre  5=  the  volume  or  capacity  of  1  kilogramme  of  distilled 
water  at  40  C.     Equals  35*196  imperial  fluid  ounces, 
decilitre    = 
centilitre  = 
millilitre 


-^  of  a  litre. 
-1-  of  a  litre. 


irnr 

Ttftnr  of  a  litre- 
cubic  centimetre  =  TTjVo  °^  a  litre. 


Factors    for    Conversion    from    One    Scale 
to  the  Other. 

Grammes  into  grains 

,,         into  ounces,  avoirdupois 
Kilogrammes  into  pounds 
Grains  into  grammes 
Avoirdupois  ounces  into  grammes 
Troy  ounces  into  grammes 
Cubic  centimetres  into  fluid  ounces,  impl 
Litres  into  fluid  ounces,  imperial 
Fluid  ounces  into  cubic  centimetres 
Pints  into  litres 
Metres  into  inches 
Inches  into  metres 

The  following  tables  give  metric  equiv 
measures  of  mass  and  capacity  :— 


.   X 

15-432 

.   X 

0*03527 

.  '  X 

2*2046 

X 

0*0648 

.   X 

28*35 

.   X 

31*104 

1.   X 

0-0352 

.   X 

35'2 

.   X 

28*42 

.   X 

0*568 

.  .  X 

39"37 

.   X 

0*0254 

alents  of  imperial 


J  8  PUBLIC     HEALTH    CHEMISTRY 

Length. — 
i  mm.  (millimetre)  =  ^  of  an  inch, 
i  cm.  (centimetre)    =  f  of  an  inch, 
i  inch  =  25*4  millimetres  or  2 J  centimetres. 

Mass. — 

1  mgr.  (milligramme)  =  0-01543  grain  (or  approx.  fc  gr.). 

rgrm.  (gramme)  =  i5'4323  grains. 

1  kgr.  (*  kilo  "  or  kilogramme)  =  2  lb.  3 \  oz.  avoirdupois. 

1  pound  avoirdupois  =  453'592  grammes. 

1  ounce  avoirdupois  =  28-35  grammes. 

1  grain  =  0*0648  gramme  or  64*8  milligrammes. 

Capacity. — 

1  centimil  =  0*17  minims  (approx.)  imperial  measure. 

1  decimil    =  1*7  minims  (approx.)  imperial  measure. 

1  c.c.  (cubic  centimetre)  (or  1  mil)  =  16*9  minims,  imperial 

1  '■-'■■'■  measure.  •; 

1  L.  (litre)  =35-196  fluid  ounces  (35  fl.  oz.,  1  fl.  dr., 
34  min.),  imperial  measure. 

1  fl.  ounce,  imperial  measure  =  28-42  cubic  centimetres 

i  pint,  imperial  measure  =  568-34  cubic  centimetres. 

1  gallon,  imperial  measure  =  4*546  litres,  or  10  lb.  avoir- 
dupois of  pure  water  at  620  F.,  and  under  an  atmos- 
pheric pressure  of  30  inches  of  mercury. 


CHAPTER    II. 

WATER    ANALYSIS. 

The  examination  of  water  samples  is  most  commonly 
made  to  determine  the  presence  or  absence  of  evidence  of 
sewage  pollution.  If  the  pollution  is  gross,  the  evidence 
of  the  unaided  senses  will  cause  its  rejection.  Few  people 
would  use  for  domestic  purposes  water  which  was  turbid, 
or  contained  suspended  matter,  or  had  a  peculiar  colour, 
or  smelt  badly,  or  had  a  disagreeable  taste.  Yet  a  water 
may  have  none  of  these  characteristics,  and  still  be 
dangerous  or  unfit  for  domestic  use.  Chemical  analysis 
is  then  often  of  service  in  distinguishing  good  from  bad 
waters.  Except  in  the  case  of  a  poisonous  metal,  the 
analysis  does  not  aim  at  finding  things  deleterious  in  them- 
selves, but  the  search  is  made  for  constituents  which 
suggest-  the  presence  of  deleterious  or  dangerous  substances. 
In  the  case  of  sewage  pollution,  the  latter  are  micro- 
organisms, and  the  former  are  those  constituents  of  sewage 
which  are  readily  detected,  namely,  chlorides  from  the 
urine,  ammonia  from  the  urea,  and  so-called  albuminoid 
ammonia  from  any  albuminous  matter.  As  the  average 
adult  excretes  6  to  9  grammes  of  chlorine  daily  as  chlorides, 
and  1  part  of  chlorine  per  100,000  parts  of  water  is  easily 
estimated,  the  pollution  produced  by  one  day's  excretion 
of  urine  into  a  water  would  thus  be  inferable  from  a  rise 
of  the  chlorine  content  1  per  100,000  even  where  the  dilu- 
tion was  into  600-900  litres  or  120-180  gallons  of  water. 
The  ammonia  estimation  is  much  more  sensitive,  a  rise  of 
1  part  in  50,000,000  being  detectable.  The  urea  excretion 
of  an  adult  averages  33  grammes  per  day,  and  by  the  influ- 
ence of  the  micrococcus  ureae  this  is  changed  to  ammonium 
carbonate,  thus  : — 

CO(NH  2)  2  +  2H  20  =  CO  3(NH  4)  2 

60  96 

Hence,  if  60  grammes  of  urea  give  rise  to  96  grammes  of 

ammonium  carbonate,  containing  34  grammes  of  ammonia, 

33  grammes  of  urea  will  give  rise  to  the  formation  of  187 


20  PUBLIC    HEALTH    CHEMISTRY 

grammes  of  ammonia,  which  diluted  into  900,000  litres  or 
200,000  gallons  of  water,  would  still  have  caused  an  appreci- 
able rise  on  the  amount  (if  any)  of  ammonia  already  present. 
This  illustrates  the  delicacy  of  some  of  these  tests. 

Various  other  estimations  are  carried  out,  such  as,  to 
determine  the  presence  or  absence  of  poisonous  metals, 
the  degree  of  hardness,  etc.  These  are  usefully  sum- 
marized thus : — 

Water  Analysis. 

This  consists  of  three  parts  : — 

1.  Physical  examination. 

2.  Chemical  examination. 

3.  Bacteriological  examination. 

Physical  Examination — 
Transparency — clear  and  bright — turbid. 
Suspended    matter — stand    for    twenty-four    hours    in 
glass  with  conical  bottom. 
Colour — two-foot  tube. 

Taste — uncertain — iron  detectable  in  I  gr.  per  gallon — 
NaCl  in  75  grains  per  gallon. 
Smell. 
Microscopic  characters  of  sediment — 

Particles  of  animal,  vegetable,  and  mineral  origin. 

Micro-organisms,  bacterial  and  protozoal. 

Chemical  Examination. — 
Reaction. 

Dissolved  solids — Total. 
Fixed. 
Volatile. 

Charring   on  ignition — fumes — odour. 
Chlorine. 

Poisonous  metals — Pb,  Cu,  Fe,  Zn,  As,  Sn. 
Lime  and  magnesia. 
Phosphates  and  sulphates. 

Free  carbonic  acid — bicarbonates — carbonates ;  dissolved 
oxygen ;  sulphuretted  hydrogen. 
Hardness — Total. 

Permanent. 
Temporary. 


WATER    ANALYSIS  21 

Free  and  saline  ammonia. 
Albuminoid  ammonia. 
Oxygen  absorption. 
Nitrates. 
Nitrites. 

Bacteriological  Examination. — 

(a)  Absolute  minimum. 

1.  Enumeration  of  bacteria  growing  in  a  medium  at  air 
temperature  i8°-22°  C. 

2.  Search  for  Bacillus  coli.  If  found — identification 
and  enumeration. 

(b)  Additional. 

3.  Enumeration  of  bacteria  growing  at  blood  heat  370  C. 

4.  Search  for  and  enumeration  of  streptococci. 

5.  Search  for  Bacillus  enteritidis  sporogenes. 

(c)  Special  procedures. 

6.  Isolation  of  Bacillus  typhosus  from  water. 

7.  Isolation  of  Spirillum  cholerae. 

COLLECTION     OF     SAMPLE. 

A  fair  average  sample  should  be  taken  in  a  clean  glass 
vessel  with  a  glass  stopper.  In  filling  the  vessel  from  a 
pond,  lake,  reservoir,  or  river,  the  mouth  of  it  should  be 
sunk  two  inches  below  the  surface,  and  the  vessel  should 
be  filled  and  emptied  once  or  twice.  If  a  surface  specimen 
is  wanted,  then  of  course  the  sample  will  be  so  taken. 
When  sampling  water  from  a  pipe  or  tap,  unless  the  effect 
of  the  water  on  the  pipes  is  under  examination,  the  water 
should  be  allowed  to  run  to  waste  for  a  few  minutes  before 
filling  the  vessel. 

The  stopper  should  be  tied  in  but  not  sealed. 

A  convenient  receiver  is  a  Winchester  quart  bottle 
which  holds  half  a  gallon,  and  this  is  a  suitable  quantity 
for  the  usual  analysis. 

Along  with  the  sample,  a  written  statement  should  be 
sent,  giving  full  particulars  as  to  mode  of  collection,  place, 
time,  recent  meteorological  conditions,  reason  why  analysis 
is  desired,  etc.,  etc. 


22  PUBLIC    HEALTH    CHEMISTRY 

The  examination  should  be  undertaken  as  soon  as  pos- 
sible, since  changes  take  place  on  keeping.  If  delay  is 
unavoidable,  changes  should  be  kept  at  a  minimum  by 
packing  in  ice. 

PHYSICAL     EXAMINATION. 

Transparency. 

Suspended  matter. 

Colour. — Best,  bluish  or  greyish  ;  greenish,  from  algae  ; 
yellow  or  brown  suspicious,  except  peaty. 

Taste. 

Smell. — Place  250  c.c.  in  a  glass-stoppered  bottle.  Put 
on  water-oven  at  300  C.  for  a  few  minutes.  Remove 
stopper,  and  smell  at  once. 

Sediment. — Let  water  stand  for  a  few  hours,  pipette  a 
few  c.c.  from  bottom,  centrifuge,  mount  a  drop  on  a 
clean  slide,  and  examine.  The  deposit  may  contain  a  very 
large  number  of  things. 

1.  Mineral  matter,  such  as  sand,  clay,  etc. 

2.  Vegetable    matter  :      (a)  Living — such    as    bacteria, 

yeasts,  moulds,  diatoms,  desmids,  rotiferae ;  (b) 
Dead — vegetable  cells,  husks  of  grain,  cotton  or 
linen  fibres,  starch  granules. 

3.  Animal  matter  :     (a)  Living — such   as   ova,   insects, 

worms,  etc.  ;  (b)  Dead — such  as  hairs,  scales, 
muscle  fibre. 

CHEMICAL     EXAMINATION. 

Reaction. — Most  drinking  waters  are  alkaline  in  reac- 
tion. Upland  surface  water  is  often  acid  from  humic  and 
ulmic  acids ;  and  this  is  important,  as  these  acids  dissolve 
lead.  Sewage-contaminated  waters  usually  retain  their 
alkalinity.  Waters  polluted  by  refuse  from  chemical 
or  dye  works  are  sometimes  acid  in  reaction. 

Dissolved  Solids. — The  suspended  matter  is  usually 
allowed  to  settle  before  testing  for  the  solids  in  solution. 
The  latter  are  estimated  as  total,  fixed,  and  volatile.  Also 
note,  -when  igniting  dried  solids,  presence  or  absence  of 
fumes,  odour,  and  charring. 

Total  Solids. — (1)  Take  a  weighed  platinum  or  porcelain 
dish  of  sufficient  size  ;     (2)  Add   100,   200,   250,   500,   or 


WATER    ANALYSIS  23 

iooo  c.c.  of  water  sample  ;  (3)  Reduce  bulk  by  moderate 
heat,  avoiding  boiling  or  spurting.  Or,  to  a  small  dish 
successive  quantities  of  the  sample  are  added,  a  note  being 
kept  of  the  amount  ;  (4)  Evaporate  to  complete  dryness 
at  ioo°  C.  (2120  F.)  on  water-bath  ;  (5)  Now  place  in 
water-oven  at  ioo°  C.  for  half  an  hour  to  remove  all  traces 
of  moisture.  Some  analysts  advise  this  drying  to  be  done 
at  1050  C.  in  hot-air  chamber  ;  (6)  Cool  in  dessicator  ; 
(7)  Weigh.  To  make  quite  sure  that  residue  is  dry,  items 
5,  6,  and  7  can  be  repeated  until  weight  is  constant  ;  (8) 
Subtract  weight  of  dish  ;  difference  is  weight  of  total 
residue  in  amount  of  sample  taken  ;  (9)  Calculate  as 
parts  per  100,000,  and  as  grains  per  gallon. 

Fixed  Solids. — (1)  Incinerate  the  dried  solids  at  as  low 
a  heat  as  possible  ;  (2)  Watch  the  process  for  blackening 
or  charring,  fumes  or  odour.  A  piece  of  dry  starch  and  KI 
paper  held  over  crucible  will  detect  any  nitric  oxide  given 
off ;  (3)  Cool  and  weigh  ;  (4)  Difference  from  weight  of 
dish  gives  fixed  solids  in  amount  of  sample  taken.  Calcu- 
late as  before. 

Volatile  Solids. — Total  solids  less  fixed  solids,  gives 
volatile.  Consist  of  organic  matter,  nitrates,  nitrites, 
ammoniacal  salts,  combined  water,  combined  carbonic 
acid,  and  sometimes  chlorides.  Should  not  exceed  1-5 
per  100,000  in  a  very  good  water. 

Example. — Evaporated  200  c.c.  sample  water  to  dryness, 
dried  in  air-oven,  cooled  in  dessicator,  and  weighed  : — 

Weight  . .  . .  . .   19-674  grammes. 

Weight  of  platinum  dish..    19*554        ,, 
Difference     . .  . .  . .     0-120     gramme. 

i.e.,  0-120  grm.  in  200  c.c.  water,  or  0-060  grm.  in  100  c.c. 
or  100  grm.,  or  60  grm.  in  100,000  grm.,  or  60  parts  per 
100,000  parts. 

Incinerated  total  residue  at  low  heat.  No  blackening, 
fumes,  odour,  nor  change  in  starch  and  KI  paper.  Cooled 
and  weighed  : — 

Weight        . .  . .  . .     19-650  grammes. 

Weight  of  platinum  dish . .     19-554 
Difference  . .  . .  . .       0-096  gramme. 


24  PUBLIC     HEALTH    CHEMISTRY 

i.e.  0-096  grm.  of  fixed  residue  in  200  c.c.  of  sample  water, 
or  0-048  grm.  in  100  c.c.  or  in  100  grm.,  or  48  parts  of 
fixed  solids  per  100,000  parts  of  sample.  Then  volatile 
solids  =  60  —  48  =  12  parts  per  100,000  parts. 

Chlorine  (present  as  Chlorides). — This  is  estimated 
by  precipitation  with  silver  nitrate — the  end  of  the  process 
being  known  by  the  use  of  a  few  drops  of  potassium 
chromate,  which  gives  a  red  precipitate  with  silver  nitrate. 
So  long  as  there  is  any  chlorine  in  solution,  however,  the 
red  precipitate  which  forms  when  each  drop  of  silver 
nitrate  solution  is  added,  is  immediately  dispelled.  The 
first  indication  of  the  red  colour  persisting  is  taken  as  the 
end  of  the  reaction. 

The  water  sample  must  be  neutral,  and  certainly  not 
acid.     It  should  also  be  colourless,  or  nearly  so. 

Solutions  required  :  (1)  5  per  cent  solution  of  potassium 
monochromate,  K  2CrO  4,  free  from  chlorine  ;  (2)  Silver 
nitrate  solution,  either  decinormal  or  standard,  say  1  c.c.  = 
1  mgr.  CI. 

Process. — (1)  Take  100  c.c.  sample  in  a  clean  porcelain 
basin  ;  (2)  Add  a  few  drops  of  chromate  solution,  which 
gives  the  liquid  a  yellow  tinge  ;  (3)  Fill  burette  with 
silver  nitrate  solution,  and  level ;  (4)  Run  in  the  solution 
drop  by  drop,  stirring  the  while  ;  (5)  Stop  when  the  least 
permanent  red  tint  is  got  ;  (6)  Calculate  amount  present 
in  parts  per  100,000,  and  grains  per  gallon. 

After  a  preliminary  trial,  the  end  reaction  can  be  more 
accurately  watched,  and  a  second  estimation  should  always 
be  done. 

Example. — 100  c.c.  sample  took  6-5  c.c.  standard  silver 
nitrate  solution,  of  which  1  c.c.  ==  1  mgr.  CI.  Therefore, 
there  are — 

6-5  X  1  =  6-5  mgr.  CI  in  100  c.c.  sample 
in  100  grm.    ,, 
in  100,000  mgr.   of  sample 
that  is,  6-5  parts  per  100,000  parts. 

For  grains  per  gallon  multiply  result  by  0-7,  thus  : — 
6-5  x  0-7  ass  4-55  grains  per  gallon. 

The   result   is   sometimes   required  in   terms   of   NaCl. 


WATER    ANALYSIS  25 

Every  molecule  of  NaCl  =  58-5  parts,  of  which  35-5  parts 
are  CI.  Therefore  one  part  of  CI  ===  58-5  -r-  35-5  =  1-65 
part  NaCl.  Then  6*5  parts  CI  per  100,000  parts  of  sample 
becomes  6-5  X  1*65=  107  parts  NaCl  per  100,000  parts 
of  sample. 

With  decinormal  silver  nitrate  solution  the  process  is 
similar,  but  as  1  c.c.  =  3-55  mgr.  of  CI,  much  less  solu- 
tion will  be  required.  On  this  account  a  larger  quantity 
of  sample  is  frequently  taken,  say  250  c.c.  When  more 
than  10  c.c.  of  standard  silver  solution  are  required  in  the 
titration,  it  is  advisable  to  repeat  the  process  after  diluting 
the  sample  water  with  distilled  water.  In  this  way  a 
more  accurate  result  is  obtained. 

The  purest  water  as  a  rule  contains  less  than  1-5  parts 
CI  per  100,000.  Increase  may  be  due  to  sea-water,  salt- 
bearing  strata,  sewage,  etc.,  and  gives  cause  for  suspicion 
until  explained  satisfactorily. 

Poisonous  Metals. — Under  this  heading  are  usually 
included  Pb,  Cu,  Fe,  and  Zn. 

The  presence  of  lead,  copper,  or  iron  in  appreciable 
amount  can  be  determined  by  taking  100  c.c.  in  a  Nessler 
glass,  and  adding  one  or  two  drops  of  ammonium  sulphide 
solution,  when  some  darkening  of  the  sample  will  occur  in 
proportion  to  the  quantity  present.  If  no  change  is  noted, 
compared  with  a  control,  then  the  sample  will  require  to 
be  concentrated,  and  tests  applied. 

A  delicate  qualitative  test  is  to  take  two  100  c.c.  Nessler 
glasses,  and  fill  one  to  the  100  c.c.  mark  with  sample  and 
the  other  with  distilled  water.  To  each  then  add  a  few 
drops  of  solution  of  permanganate  of  potash  to  give  them 
a  distinct  pink  tinge.  Then  add  to  each  1  c.c.  of  sulphuric 
acid  and  1  c.c.  of  potassium  ferrocyanide,  and  compare  the 
tints. 

Iron  gives  a  blue  tint  ;  copper  gives  a  brown  ;  zinc 
gives  a  white  ;  and  lead  gives  no  change.  The  control 
shows  no  change. 

Qualitative  Test  Table. — Concentrate  sample  to  one- 
fifth  of  its  bulk,  say  200  c.c.  to  40  c.c,  and  test  thus  : — 

To  5  c.c.  in  a  test  tube  add  a  few  drops  of  Am  2S  solution. 
Black  precipitate  may  be  lead,  copper,  or  iron. 
White  ,,  ,,       zinc. 


26      PUBLIC  HEALTH  CHEMISTRY 

i.  If  precipitate  is  black,  divide  into  two  portions. 

a.  To  one  portion  add  dilute  HC1 ;  if  precipitate  dis- 
solves, then  iron  is  present.  Confirm  for  iron.  Take  two 
tubes  with  5  c.c.  of  concentrated  sample  in  each,  after 
heating  for  a  few  minutes  with  a  pinch  of  potassium 
chlorate  to  oxidize  the  iron  to  the  ferric  state.  To  one, 
add  solution  of  KCNS ;  blood-red  colour  produced.  To 
other,  add  solution  of  K  4FeCy  6  ;  prussian  blue  colour. 

b.  To  other  portion  add  KCN  ;  if  precipitate  dissolves, 
copper  present.  Confirm  for  copper.  As  above,  take 
two  tubes  with  concentrated  solution,  but  without  treating 
in  any  way.  To  one,  add  AmOH  ;  blue  colour.  To  other, 
add  K4FeCy6  ;    bronze  precipitate. 

c.  If  precipitate  does  not  dissolve  ;  then  lead.  Confirm 
for  lead.  As  above,  take  two  tubes  with  concentrated 
solution.  To  one  add  KI  solution  ;  yellow  precipitate, 
soluble  on  boiling.  To  other  add  K  2CrO  4  solution  ; 
yellow  precipitate,  soluble  in  KOH. 

2.  If  precipitate  is  white,  confirm  for  zinc.  Take  two 
tubes  as  before.  To  one,  add  AmCl,  AmOH  and  Am  2S  ; 
white  precipitate.  To  other,  add  K4FeCy6;  white 
gelatinous  precipitate. 

Arsenic. — Take  a  litre  of  the  water  sample,  add  pure 
sodium  carbonate  until  alkaline,  and  evaporate  nearly  to 
dryness.  Test  concentrated  liquid  by  Reinsch's  or  Marsh's 
test.     The  former  is  described  under  Beer  (page  129). 

Tin. — Evaporate  a  litre  of  water  sample  to  dryness,  ash 
the  residue;  exhaust  ash  repeatedly  with  strong  HC1,  evapor- 
ate portions  to  dryness  on  water-bath,  add  some  water, 
boil,  and  filter.  Test  filtrate  with  H  2S  ;  a  dingy,  yellow 
precipitate,  soluble  in  Am2S,  indicates  the  presence  of  tin. 
The  precipitate  is  also  soluble  in  the  caustic  alkalies. 

Quantitative  Estimation. — This  depends  on  a  colori- 
metric  method.  100  c.c.  of  the  sample  water  are  taken  in  a 
Nessler  glass,  and  a  measured  quantity  of  a  suitable  precipi- 
tating agent  is  added.  According  to  the  amount  of  metal 
present,  a  certain  depth  of  coloration  is  obtained.  100  c.c. 
of  distilled  water  are  now  taken  in  a  Nessler  glass,  and  the 
same  quantity  of  precipitant  added  to  them.  From  a  burette, 
successive  small  quantities  of  a  standard  solution  of  the. 
metal  being  tested  for  are  added  to  the  glass  containing 


WATER    ANALYSIS  27 

the  distilled  water  ;  and  after  each  addition  the  coloration 
produced  is  compared  with  that  in  the  glass  containing  the 
sample  water.  This  is  done  by  putting  both  glasses 
together  on  a  white  tile,  or  on  a  Nessler  stand,  and  looking 
down  through  the  liquids.  If  the  tints  are  alike  in  depth, 
then  the  glasses  have  been  matched.  If  the  sample  is  the 
darker,  more  standard  will  require  to  be  added  to  the  other 
glass,  and  a  further  comparison  made.  If  the  sample  is 
the  lighter,  a  fresh  amount  of  distilled  water  will  require 
to  be  put  up,  and  less  standard  solution  added  at  first.  In 
any  case  the  comparisons  are  continued  until  matching  of 
the  tints  is  obtained,  and  then  the  amount  of  standard 
solution  which  has  been  added  to  the  distilled  water  is 
held  to  measure  the  amount  of  metal  present  in  the  ioo  c.c. 
of  sample  water. 

The  process  of  comparing  the  tints  or  colorations  to  a 
match  is  called  "  Nesslerizing,"  and  will  frequently  be  used 
in  subsequent  work. 

Determination  of  Lead. — A  standard  solution  of  lead 
acetate  is  required,  of  such  a  strength  that  i  c.c.  =  o-i 
mgr.  (TV  mgr.)  of  lead.  Pb  (C2H302)2  +  3H20;  the 
molecular  weight  =  379,  and  contains  207  parts  of  lead. 
Therefore  379/207  ==  1-83  parts  of  lead  acetate  contain 
1  part  of  lead.  That  is,  1-83  grm.  of  lead  acetate  contain 
1  gram,  of  lead.  If  we  dissolve  1-83  grm.  of  lead  acetate 
in  1  litre  of  distilled  water,  1  c.c.  will  contain  1  mgr.  of 
lead.  This  solution  diluted  ten  times  gives  a  standard 
solution  of  lead  acetate  such  that  1  c.c.  =  o-i  mgr.  Pb. 
A  solution  of  Am2S  in  water  is  also  required. 

Process. — Take  two  100  c.c.  Nessler  glasses,  with 
distinctive  marks  affixed.  To  one  add  100  c.c.  of  sample 
water.  To  the  other  add  100  c.c.  distilled  water.  To 
both  add  2  c.c.  of  Am2S  solution,  and  stir.  Now  take  a 
burette,  and  fill  it  with  the  standard  solution  of  lead 
acetate.  Add  1  c.c.  of  this  to  the  distilled  water  and  stir. 
Compare  coloration  produced  with  that  in  glass  containing 
sample.  If  the  sample  is  darker,  add  another  c.c.  of 
standard  solution  to  the  other  glass,  stir,  and  compare. 
Repeat  procedure  until  a  match  is  obtained.  If  the  sample 
is  lighter  than  the  coloration  produced  by  1  c.c.  of  standard 
solution,  begin  again  and  add  -|  c.c.  of  standard  solution. 


28  PUBLIC    HEALTH    CHEMISTRY 

Example. — Suppose  3  c.c.  of  standard  solution  were 
required  to  match  sample.  1  c.c.  of  standard  solution  lead 
acetate  =  o-i  mgr.  Pb.  Therefore  3  c.c.  of  standard  solu- 
tion lead  acetate  =  0-3  mgr.  Pb.  Hence  there  is  0-3  mgr. 
Pb  in  100  c.c.  of  sample  water,  or  0*3  mgr.  Pb  in  100  grm. 
of  sample  water,  or  0-3  mgr.  Pb  in  100,000  mgr.  of  sample 
water  ;  that  is,  0-3  part  of  Pb  in  100,000  parts  of  sample 
water.  By  this  method  0-05  part  per  100,000,  or  JF  gr. 
per  gallon,  may  be  easily  detected. 

Many  waters,  especially  soft  and  peaty  waters,  possess 
a  coloration  sufficient  to  equal  that  produced  by  0-5  c.c., 
or  even  1  c.c.  of  standard  lead  solution.  In  such  a  case, 
carefully  match  the  natural  coloration  in  terms  of  the 
standard  solution,  and  deduct  the  amount  obtained  from 
the  amount  required  in  the  regular  test.  Where  the 
coloration  is  deeper  still,  and  a  poisonous  metal  is  sus- 
pected, evaporate  100  c.c.  to  dryness,  ignite  to  get  rid  of 
vegetable  colouring  matter,  digest  the  ash  with  HC1,  filter, 
collect  filtrate,  washing  filter-paper  with  distilled  water, 
and  make  up  bulk  of  filtrate  to  100  c.c.     Now  test  as  before. 

As  lead  is  a  cumulative  poison,  its  presence  in  a  water 
should  disqualify  that  water  for  domestic  use. 

Copper. — Copper  is  similarly  estimated,  using  a  standard 
solution  of  copper  sulphate,  CuSO  4  +  5H  20.  The  mole- 
cular weight  of  this  salt  in  crystals  is  249,  and  as  this 
amount  contains  63  parts  of  copper,  249/63  or  3-95  parts 
will  yield  1  part  of  copper.  Hence  3-95  grm.  of  copper 
sulphate  crystals  dissolved  in  1  litre  of  distilled  water 
gives  a  solution  such  that  1  c.c.  contains  1  mgr.  of  copper. 
This  diluted  ten  times  gives  the  desired  standard  solution, 
1  c.c.  =  o-i  mgr.  Cu. 

Copper  can  also  be  estimated  by  precipitation  with  HC1 
and  potassium  ferrocyanide,  which  gives  a  bronze  color^ 
ation. 

Iron. — Iron  is  best  estimated  by  oxidizing  it,  if  necessary, 
to  the  ferric  state,  and  then  adding  potassium  sulpho- 
cyanate,  which  produces  a  blood-red  colour.  Reagents 
required  :  (1)  Standard  solution  of  ferrous  sulphate, 
FeSO  4  +  7H  20,  0-496  grm.  dissolved  in  1  litre  of  distilled 
water  (acidified  with  H2S04),  1  c.c.  of  this  solution  = 
0'i   mgr.    Fe.      Dilute  ten  times   in   use,  then   1    c.c.  = 


WATER    ANALYSIS  29 

o-oi  mgr.  Fe  ;  (2)  Dilute  HNO  3  solution  :  make  up 
30  c.c.  of  pure  concentrated  acid  to  100  ex.  with  distilled 
water  ;  (3)  Potassium  sulphocyanate  solution  :  15  grm. 
KCNS  dissolved  in  100  c.c.  of  distilled  water. 

Process. — To  each  of  two  Nessler  glasses  add  5  c.c.  of 
each  solution  (2)  and  (3).  Add  1  c.c.  of  standard  solution 
to  one,  and  to  the  other  add  a  measured  quantity  of 
sample,  say  10  c.c,  and  note  depth  of  tint  produced, 
compared  to  standard.  If  the  two  are  near  each  other, 
proceed  jas  before  to  match.  If  the  sample  is  much  too 
dark,  use  less  ;  if  much  too  light,  use  more.  Always  make 
up  bulk  in  each  Nessler  to  be  approximately  equal  to  each 
other  by  adding  distilled  water,  before  finally  matching. 
If  more  than  3  c.c.  of  standard  solution  are  required,  the 
tint  gets  too  deep. 

Iron  is  perceptible  to  taste  when  present  to  the  extent 
of  I  gr.  per  gallon,  or  1  part  in  350,000  parts  of  water. 

Zinc  is  usually  determined  gravimetrically.  It  may  be 
done  volumetrically  with  standard  solution  of  potassium 
ferrocyanide,  using  copper  sulphate  as  an  indicator,  the 
brown  copper  precipitate  not  being  formed  until  all  the  Zn 
is  precipitated.  The  standard  solution  is  made  0-324  grm. 
of  K4Fe(CN)  6 .  3H  20  per  litre.     Then  1  c.c.  =  o-i  mgr.  Zn. 

Lime  and  Magnesia. — These  are  often  present  together 
from  strata. 

Lime. — Ammonium  oxalate  gives  a  turbidity  with  9 
parts  per  100,000,  and  a  white  precipitate  with  anything 
over  20  parts  per  100,000. 

Magnesia. — Precipitate  any  lime  present  with  ammonium 
oxalate,  filter,  then  add  AmCl,  AmOH,  and  sodium  phos- 
phate. Crystals  of  so-called  triple  phosphate  will  separate 
out  in  twenty-four  hours.     (MgNH4P04.) 

For  this  and  the  next  test  it  is  better  to  concentrate 
to  «V 

Phosphates. — Add  some  dilute  nitric  acid,  stir,  add 
excess  of  ammonium  molybdate,  and  heat.  If  phosphates 
are  present,  a  yellow  colour  will  form. 

Sulphates.  —  Add  dilute  HC1  and  barium  chloride 
solution — a  white  precipitate  of  sulphate  of  barium,, 
insoluble  in  all  acids. 


30      PUBLIC  HEALTH  CHEMISTRY 

Quantitative  Estimation  of  Lime,  Magnesia, 
Phosphates,  and  Sulphates. 

These  are  all  estimated  for  gravimetrically  by  precipita- 
ting as  above,  collecting  the  precipitate,  which  settles 
after  twelve  hours,  on  a  filter-paper  of  known  ash  ;  drying, 
and  igniting ;  and  weighing  ash.  The  ash  of  lime  is  weighed 
as  calcium  carbonate,  of  magnesia  as  magnesium  pyro- 
phosphate (Mg2P207),  of  sulphates  as  barium  sulphate. 
The  phosphates  are  precipitated  as  triple  phosphates  of 
magnesium  and  weighed  similarly.  In  the  case  of  magne- 
sium, the  lime  salts  must  be  removed  before  precipitating, 
hence  the  filtrate  from  the  lime  estimation  is  suitable  for 
the  purpose. 

GASES     IN    WATER. 

Carbon  Dioxide  in  Water. — Exists  as  free  CO  2 ;  bicar- 
bonate and  carbonate  ;  and  free  CO  2  and  bicarbonate. 

i.  Free  C02. — Determined  by  titration  with  N/20 
sodium  carbonate  solution,  using  phenolphthalein  as  indi- 
cator. On  adding  the  N/20  solution  from  a  burette  to 
the  sample,  a  red  colour  appears  which  gradually  fades  as 
the  carbonate  absorbs  the  free  carbon  dioxide  and  changes 
to  bicarbonate,  which  is  neutral  to  phenolphthalein. 

Na2C03  +  C02  +  H20  =  2NaHC03 
106  44 

N/i  sod.  carb.  =  53  grm.  per  litre  and  absorbs  22 
grm.  carbon  dioxide  per  litre  ;  hence  N/20  sod.  carb. 
=  2-65  grm.  per  litre  and  absorbs  i-i  grm.  carbon 
dioxide  per  litre;  hence  1  c.c.  N/20  will  absorb  o-oon 
grm.  free  C02. 

Process. — Take  100  c.c.  of  sample  in  an  Erlenmeyer 
flask  on  a  white  slab,  and  add  one  drop  of  1  per  cent 
alcoholic  solution  of  phenolphthalein. 

Fill  burette  with  N  /20  solution  of  sodium  carbonate  and 
add  to  sample,  c.c.  by  c.c,  until  a  permanent  red  tint 
remains  after  waiting  a  minute  or  two. 

Calculate  out  result. 

Free  carbon  dioxide  is  almost  constantly  present  in 
ground  waters,  and  in  inverse  ratio  to  the  amount  of 
dissolved  oxygen.     It  may  be  as  high  as   13   parts  per 


WATER    ANALYSIS  31 

100,000,  probably  derived  irom  ground  air,  increasing  with 
the  depth  and  decreasing  with  the  porosity  of  the  soil. 

2.  Carbonate  and  Bicarbonate. — The  carbonate  is  first 
determined  by  titration  oi  the  sample  with  the  standard 
solution  of  oxalic  acid,  in  presence  of  phenolphthalein, 
which  gives  a  pink  colour  with  carbonates  and  alkalies,  and 
is  colourless  with  bicarbonates  and  acids. 
H  2C  20  42,H  20+2Na  2C0  3  =*=  2NaHC0  8+Na  2C  20  4+2H  20 

90  +  36       2  x  106 

From  the  equation  we  see  that  126  grim  of  crystallized 
oxalic  acid  react  with  212  grm.  of  sodium  carbonate, 
containing  88  grm.  of  C02,  and  change  it  to  sodium 
bicarbonate.  Thus  126  grm.  of  oxalic  acid  crystals 
measure  the  change  of  88  grm.  of  G02  from  the  state  of 
carbonate  to  that  of  bicarbonate,  or  126/88  =  1-43  grm. 
measure  the  change  in  1  grm.  of  C02.  Hence  a  standard 
solution  of  oxalic  acid  crystals,  1-43  grm.  per  litre,  is  of 
such  a  strength  that  1  litre  ==  1  grm.  CO  2,  or  1  c.c.  =  imgr. 
C02.  When  all  the  carbonate  is  changed  to  bicarbonate,  the 
solution  becomes  colourless,  and  the  number  of  c.c.  used 
measures  the  carbonate  present.  Boil  the  fluid  in  the  flask 
briskly  for  ten  minutes,  when  all  the  bicarbonate,  whether 
originally  present  or  derived  from  carbonate,  is  decom- 
posed with  formation  of  carbonate  (signalized  by  the  return 
of  the  pink  colour)  and  evolution  of  C02,  as  shown  thus  : — 

2NaHC0  3  -  Na  2C0  3  +  H  20  +  CO  2 
Cool  and  repeat  the  titration  with  the  standard  oxalic 
solution  ;  the  number  of  c.c.  required  measures  the 
amount  of  carbonate  now  in  the  sample.  But  this  figure 
represents  only  half  the  C02  present  before  boiling,  halt 
of  the  CO  2  having  gone  into  the  atmosphere.  Hence  the 
figure  has  to  be  doubled,  and  then  measures  the  total 
amount  of  C02  present  originally  in  the  sample.  The 
amount  present  as  carbonate  is  known  from  the  first 
titration,  and  so  the  amount  present  as  bicarbonate  is 
measured  by  the  difference  between  the  number  of  c.c. 
used  in  the  first  titration,  and  double  the  number  used  in 
the  second  titration.     The  rationale  may  be  shown  thus : — 


32  PUBLIC    HEALTH    CHEMISTRY 

(2),  NaHC08+    j  fi      b  u  d=N     CQ  +R  0+co 

(3).  2Na 2C0 3+H 2C ,0 4+Phth=2NaHC0 3+Na 2C 20 4 

Example  :  ioo  c.c.  of  sample,  plus  phth.,  required  8  c.c. 
of  std.  oxalic  sol.  to  decolorize.  Boiled ;  cooled  ;  and 
titrated  again,  when  n  c.c.  were  required. 

COo  as  Carbonate,  8  parts  per  100,000. 

CO._,  as  Bicarbonate,  22  —  8  =  14  ,,  ,, 

3.  Free  Carbon  Dioxide  and  Bicarbonate. — These  are 
estimated  by  the  addition  of  excess  of  alkali  in  the  shape 
of  a  known  quantity  of  baryta  solution.  The  baryta 
fixes  the  free  carbon  dioxide  and  that  half  bound  in  the 
bicarbonates,  precipitating  both  as  barium  carbonate. 
The  excess  of  alkali  which  remains  unused  is  measured  by 
titration  with  standard  oxalic  acid  solution. 

C02  +  BaH202  a*  BaC03  +  H20 
CaCO  3C0  2     +  BaH  20  2  =  BaCO  8  +  CaCO ,  +  2H  20 
H2C204  -f  BaH202  =  BaC204  +  2H26 

From  these  equations  we  see  that  one  molecule  of  oxalic 
acid  and  one  molecule  of  C02  are  each  able  to  neutralize 
one  molecule  of  baryta  in  solution.  Therefore,  126  grm. 
of  crystallized  oxalic  acid  neutralizes  171  grm.  of  baryta, 
which  fixes  44  grm.  of  C02;  or  126/44  =  2-86  grm.  of 
oxalic  neutralizes  171/44  grm.  of  baryta,  which  fixes 
44/44  =  1  grm.  CO  2.  Hence  a  standard  solution  of  oxalic 
acid  2-86  grm.  per  litre  is  such  that  1  c.c.  measures  the 
quantity  of  baryta  which  fixes  1  mgr.  of  CO  2. 

Process.— To  100  c.c.  of  sample  in  a  flask  or  bottle, 
add  a  drop  of  phenolphthalein,  and  then  5  c.c.  of  BaCl2 
solution,  5  c.c.  of  AmCl  solution,  and  40  c.c.  of  baryta 
solution  (0*5  per  cent).  If  excess  of  baryta  is  present, 
the  liquid  should  become  red,  and  remain  so.  Stopper 
the  vessel  and  set  aside  for  twelve  hours.  Thereafter 
titrate  the  whole  sample  (or  an  aliquot  part)  with 
standard  oxalic  acid  solution.  Titrate  40  c.c.  of  fresh 
baryta  solution.  The  difference  between  the  number 
of  c.c.  required  for  the  fresh  baryta  solution  and  that 
required  for  the  baryta  mixed  with  sample,  measured 
in  c.c.  of  standard  oxalic  solution  the  amount  of  baryta 
used   up   in   fixing   free   C02   and   changing   bicarbonate 


WATER    ANALYSIS  3a 

to  carbonate.  But  as  each'  c.c.  of  oxalic  equals  I  mgr. 
of  CO  2,  then  the  number  of  them  gives  the  total  number 
of  mgr.  of  CO  2  in  the  water  sample.  The  free  C02  is 
determined  as  before  by  N/20  sodium  carbonate  solution, 
and  the  difference  gives  the  amount  present  as  bicarbonate. 

Dissolved  Oxygen. — Winkler,  Dibdin,  Thresh,  and  Mohr 
have  all  devised  methods  for  determining  the  amount  of 
oxygen  dissolved  in  water  samples.  Any  such  method  must 
be  simple,  speedy,  and  accurate,  and  the  water  must  not 
be  operated  on  in  an  inert  atmosphere,  or  there  might  be 
a  rapid  loss  by  diffusion.  Winkler's  is  perhaps  the  most 
simple  and  readily  applied,  and  needs  no  special  apparatus. 
The  following  solutions  are  required :  (a)  Manganous 
chloride  solution  free  from  iron  (80  grm.  MnCl  2  +  4-H  20 
in  100  c.c.  of  distilled  water  ;  (b)  KI  and  NaOH  solution 
(10  grm.  KI  in  100  c.c.  of  33  per  cent  NaOH).  This 
solution  when  diluted,  and  sulphuric  and  starch  solution 
added,  should  not  give  any  blue  colour  ;  (c)  N/100  iodine 
(1-27  grm.  I  and  2  grm.  KI  dissolved  in  1  litre).  This  is 
used  to  standardize  thiosulphate  ;  (d)  N/100  thiosulphate 
of  soda  solution  (2-48  grm.  Na2S203  -f  5H20  per  litre) ; 
(e)  Starch  solution. 

Process. — Take  a  glass  bottle  provided  with  a  well- 
fitting  glass  stopper  and  of  about  300  c.c.  capacity. 
Determine  accurately  the  capacity  when  stoppered.  Wash 
it  out  with  some  of  the  water  to  be  examined,  and  then 
fill  it  to  overflowing  with  sample  water  (avoid  splashing). 
Introduce,  by  different  pipettes,  1  c.c.  of  each  of  solutions 
(a)  and  (b),  doing  this  carefully  so  that  they  are  delivered 
close  to  the  bottom  of  the  bottle.  Put  in  the  stopper 
tightly,  enclosing  no  air  bubbles.  Mix  the  contents  by 
lightly  swinging  the  bottle.  A  precipitate  forms  which  is 
allowed  to  settle.  This  takes  a  variable  time;  usually 
fifteen  minutes  is  sufficient.  When  it  has  settled  and  the 
upper  part  of  the  fluid  is  clear,  introduce  by  pipette,  so  as 
to  fall  on  to  the  precipitate,  5  c.c.  of  strong  HC1,  replace 
stopper  and  swing  until  precipitate  dissolves,  when  the 
fluid  becomes  yellow-coloured  from  liberated  iodine.  The 
contents  of  the  bottle  are  now  poured  into  a  clean  beaker, 
the  bottle  washed  out  with  distilled  water,  and  the  wash- 
ings added.     It  is  then  titrated  with  the  N  /ioo  thiosulphate 

3 


34  PUBLIC    HEALTH    CHEMISTRY 

solution,  every  c.c.  of  which  used  equals  o- 00008  grm.  O, 
or  0-055825  c.c.  oxygen.  Starch  solution  is  used  for  the 
end  reaction.  The  amount  of  oxygen  found  is  present  in 
the  capacity  of  the  bottle,  less  the  2  c.c.  of  solutions  added. 
The  result  is  returned  in  parts  by  weight  per  100,000,  or  in 
cubic  centimetres  per  litre. 

1.  2MnCla  +  4NaOH  =  4NaCl  -f  2Mn(0H)  2. 

2.  2Mn(0H)  2  +  O  +  H20  =  2Mn(0H)  3. 

3.  2Mn(OH)  3  +  6HC1  =  2MnCl3  +  6H  20. 

4.  2MnCl3  +  2KI  =  2MnCl2  +  2KCI  +  I2. 

The  process  must  be  done  rapidly.  Nitrites  liberate 
iodine  and  so  vitiate  the  result,  increasing  it.  Much 
organic  matter  interferes  with  the  method,  as  it  absorbs 
the  liberated  iodine,  thus  diminishing  the  result.  Rapid 
working  diminishes  the  latter  interference. 

The  amount  of  dissolved  oxygen  in  a  water  is  influenced 
mainly  by  temperature,  being  less  in  summer  and  more  in 
winter.  Ordinary  tap  water  in  this  country  contains  on 
an  average  7  c.c.  per  litre,  which  is  about  1  part  by  weight 
in  100,000.  Water  is  saturated  at  50  C,  io°  C,  150  C,  and 
20°  C.  respectively,  by  8-68  c.c,  777  c.c,  6-96  c.c,  and 
6-28  c.c  per  litre. 

Suphuretted  Hydrogen. — This  is  estimated  by  titration 
with  N  /ioo  iodine,  which  is  decolorized  by  the  H  2S  ;  thus 
H2S  +  I2  =  2HI  +  S. 

Process. — Take  10  c.c  N/100  iodine  in  a  white  porcelain 
basin.  Fill  a  burette  with  sample,  and  add  to  basin  until 
colour  is  gone,  using  starch  for  end  reaction.  The  N/100 
iodine  is  made  as  above,  and  is  standardized  against  N/100 
thiosulphate  solution.  Every  c.c.  of  N/100  I  equals  1  c.c.  of 
N/100  H  2S.  But  N/i  H  2S  is  17  grm.  per  litre,  therefore 
1  c.c.  N/i  =  0-017  grm->  and  1  c.c  N/100  =  0-00017  grm. 
H2S,  or  0-17  mgr.  Hence  the  number  of  c.c  of  N/100I 
used  x  0-17,  gives  the  number  of  mgr.  of  H  2S  in  the  amount 
of  sample  run  in  from  burette  to  decolorize  the  iodine. 

HARDNESS    (TOTAL,   TEMPORARY,    OR   PERMANENT). 

Hardness  is  due  to  the  presence  in  a  water  of  metallic 
salts  which  form  insoluble  compounds  with  the  fatty  acids 
usually  present  in  soap.     A  soap  is  the  oleate,  stearate, 


WATER    ANALYSIS  35 

or  palmitate  of  sodium  or  potassium.  Hard  soap  has 
soda  for  its  base,  and  soft  soap  has  potash  for  its  base. 
These  soaps  are  soluble  in  water  and  form  a  lather  there- 
with on  shaking.  When  soap  is  used  with  a  water  in 
which  lime,  magnesia,  baryta,  iron,  alumina,  or  other  such 
substances  are  present,  oleates,  etc.,  of  these  bases  are 
formed,  which  being  insoluble  are  precipitated,  and  no 
lather  can  be  produced  until  an  excess  of  soap  is  present. 
A  certain  amount  of  the  hardness  is  removable  by  boiling, 
and  this  is  called  temporary  hardness,  and  is  chiefly  due 
to  the  carbonates  of  lime  and  magnesia  held  in  solution 
by  carbonic  acid  gas,  and  by  sulphates  of  these,  with  salts 
of  silica,  alumina,  and  iron  when  present.  The  permanent 
hardness,  or  what  still  remains  in  solution  after  boiling, 
consists  mainly  of  some  sulphates,  chlorides,  and  nitrates 
of  calcium  and  magnesium,  with  a  little  iron  and  alumina. 
Free  carbonic  acid  gas  in  water  also  consumes  soap,  two 
equivalents  uniting  with  one  of  soap  as  ordinarily  estimated. 
Amount  of  hardness  is  expressed  as  grains  per  gallon 
(Clark's  degrees),  or  parts  per  100,000  (metrical  degrees) 
in  terms  of  calcium  carbonate.  In  Germany  the  hardness 
is  expressed  as  metrical  degrees  of  CaO  per  100,000. 

The  total  hardness  of  a  water  should  not  exceed  30  parts 
per  100,000,  if  for  domestic  purposes.  Hard  waters  vary 
from  20  to  30  degrees  on  the  metrical  scale  ;  a  soft  water 
from  8  to  15  ;  and  a  very  soft  water  from  8  downwards. 
The  greater  the  permanent  hardness,  the  more  objectionable 
is  the  water  ;  and  of  a  good  water  it  should  not  exceed  50 
metrical,  or  30  to  40  Clark. 

Determination  of  Hardness. — 

1.  By  Standard  Soap  Solution  Method. 

Dissolve  10  grm.  of  castile  or  soft  soap  in  1  litre  of  a 
mixture  of  equal  parts  of  distilled  water  and  methylated 
spirit.  Standardize  the  solution  so  that  1  c.c.  completely 
precipitates  1  mgr.  of  calcium  carbonate  or  an  equivalent 
salt.  The  CaC03  may  be  dissolved  in  the  least  possible 
quantity  of  HC1,  then  evaporated  to  dryness  twice,  to  get 
rid  of  the  HC1,  and  then  the  resulting  CaCl2  dissolved  in 
the  proper  amount  of  distilled  water ;  that  is,  1  grm. 
of  the  carbonate  is  treated  as  above,  and  the  resulting 


36  PUBLIC    HEALTH    CHEMISTRY 

chloride  dissolved  in  i  litre  of  aq.  dest.,  then  I  c.c.  = 
I  mgr.  CaCOg.  The  soap  solution  is  then  tested  with 
50  c.c.  of  aq.  dest.  (recently  boiled  to  get  rid  of  CO  2),  to 
determine  how  much  of  it  is  required  to  produce  a  perma- 
nent lather  ;  that  is,  a  lather  which  remains  as  a  uniform 
film  J  in.  thick  on  the  surface  of  the  water  five  minutes 
after  the  bottle  has  been  laid  on  its  side.  The  soap  solution 
is  added  from  a  burette,  y1^  c.c.  at  a  time.  The  50  c.c.  are 
contained  in  a  stoppered  bottle,  150  c.c.  capacity,  which 
is  well  shaken  after  each  addition,  then  laid  on  its  side, 
and  the  character  of  the  lather  noted.  If  it  quickly 
disappears,  then  more  soap  solution  is  added,  until  the 
lather  has  the  permanence  described.  The  amount  usually 
required  by  50  c.c.  of  aq.  dest.  varies  from  0-2  c.c.  to 
0-6  c.c,  and  should  be  determined  not  once  for  all,  but 
at  intervals,  as  it  will  vary  with  the  strength  of  the  soap 
solution,  and  this  latter  tends  to  deteriorate  on  keeping. 

The  soap  solution  is  now  standardized  against  the 
standard  CaC03  solution,  5  c.c.  of  which  are  added  to 
45  c.c.  of  recently  boiled  aq.  dest.,  contained  in  a  glass- 
stoppered  bottle  of  about  150  c.c.  capacity.  The  soap 
solution  ,is  now  added  from  a  burette,  1  c.c.  at  a  time. 
Shake  briskly  after  each  addition.  When  the  proper 
lather  is  formed,  the  shaking  of  the  bottle  produces  a  soft 
sound  which  is  different  from  the  hard  sound  at  first 
emitted,  and  heard  when  the  bottle  is  held  near  the  ear. 
Say  that  4  c.c.  of  standard  soap  solution  were  required  to 
produce  the  permanent  lather,  and  that  0-5  c.c.  were 
necessary  for  50  c.c.  of  aq.  dest.,  then  4  —  0-5  =  3*5  c.c. 
have  been  used  in  precipitating  the  5  mgr.  of  CaCOg. 
contained  in  the  5  c.c.  of  standard  calcium  solution.  But 
we  wish  the  standard  soap  solution  to  be  5  c.c.  =  5  mgr. 
or  1  c.c.  =  1  mgr.  calcium  carbonate.  Hence  it  is  too 
strong,  and  we  must  dilute  it  (with  a  mixture  of  equal 
parts  of  methylated  spirit  and  aq.  dest.)  so  as  to  make 
every  3*5  c.c.  up  to  5  c.c,  or  every  35  c.c.  up  to  50  c.c. 
The  solution  is  now  of  standard  strength,  but  requires  to 
be  re-standardized  at  intervals,  as  it  is  somewhat  unstable. 

The  standardizing  can  also  be  done  against  a  standard 
solution  of  Ba(N03)2  which  has  a  molecular  weight  of 
261  compared  to  100  for  CaC03.     Therefore  if  2*61  grnu 


WATER    ANALYSIS  37 

of  barium  nitrate  be  dissolved  in  I  litre  of  aq.  dest.  I  c.c.  = 
i  mgr.  Ba(N03)  2  equal  to  I  c.c.  =  I  mgr.  CaC03. 

Total  Hardness. — Take  50  c.c.  of  sample  in  bottle  and 
test  with  standard  soap  solution  as  above  until  a  permanent 
lather  is  obtained.  Deduct  0-5  c.c.  (say)  necessary  to 
produce  lather,  and  double  the  answer  gives  the  total 
hardness  in  metrical  degrees.  This  multiplied  by  07 
gives  it  in  grains  per  gallon,  or  Clark's  degrees.  If  more 
than  8  to  9  c.c.  be  required,  it  is  advisable  to  dilute 
25  c.c.  of  the  sample  with  25  c.c.  recently  boiled  aq.  dest., 
and  redetermine  the  hardness. 

Permanent  or  Fixed  Hardness. — Take  100  c.c.  of  sample 
water  and  make  up  to  200  c.c.  with  aq.  dest.  Boil  down 
to  one-half  its  bulk,  and  a  little  more.  Allow  to  cool  to 
6o°  F.  (15-5°  C),  and  make  up  to  100  c.c.  with  aq.  dest. 
Remove  50  c.c.  and  determine  hardness  as  before. 

By  boiling,  all  the  free  and  half-bound  CO  2  is  driven  off, 
and  nearly  all  the  CaC03  is  precipitated.  The  CaS04 
and  the  CaCl2  are  not  affected  if  the  evaporation  is  not 
carried  too  far.  Some  of  the  MgCO  3  at  first  thrown  down 
is  redissolved  as  the  water  cools. 

Temporary  or  Removable  Hardness. — This  is  the  difference 
between  the  total  and  the  fixed  hardnesses. 

Notes. — The  lime  salts  precipitate  at  once  with  the 
soap  solution  ;  the  magnesium  salts*  precipitate  slowly  ; 
hence  it  sometimes  happens  that  all  the  Ca  is  precipitated 
and  a  lather  formed  which  shortly  disappears,  and  more 
soap  solution  is  needed.  The  presence  of  magnesium  salts 
is  said  to  cause  the  lather  to  be  brown  in  colour  and  to 
break  very  easily.  More  soap  is  required  to  produce  a 
lather  with  a  certain  amount  of  magnesium,  than  with 
the  equivalent  amount  of  Ca,  though  this  is  ignored  in 
practice. 

2.  By  Hehner's  Alkalimetry  Method. 

Temporary. — Titrate  50  or  100  c.c.  of  sample  with 
N/50  H2S04,  using  methyl-orange  as  indicator,  until  a 
permanent  pink  is  got.  The  sulphuric  acid  decomposes  the 
CaCO  3  with  evolution  of  CO  2,  and  until  all  the  carbonate 
is  decomposed,  no  sulphuric  is  free  to  attack  the  methyl- 
orange.     The  number  of  c.c.  of  sulphuric  gives  the  number 


38  PUBLIC    HEALTH    CHEMISTRY 

of  mgr.  of  calcium  carbonate  in  the  amount  of  sample 
taken,  because  I  c.c.  N/50  H2S04  =  1  c.c.  N/50  CaC03. 
But  the  molecular  weight  of  CaC03  is  10O  and  N/50  = 
1  grm.  per  litre,  hence  1  c.c.  =  1  mgr.  CaC03. 

Permanent. — To  a  fresh  lot  of  sample  add  sufficient 
N/50  Na2C03  to  precipitate  as  carbonate  all  the  Ca  and 
Mg  present,  noting  carefully  amount  used. 

Evaporate  mixture  to  dryness  on  water-bath. 

Dissolve  soluble  part  of  residue  in  10  to  20  c.c.  of  aq. 
dest.  and  filter  through  small  filter-paper.  Wash  out  dish 
and  filter-paper  with  a  little  more  distilled  water  to  ensure 
complete  removal  of  all  the  Na2C03. 

Titrate  nitrate  with  N/50  H2S04,  using  methyl-orange 
as  indicator,  until  a  permanent  pink  colour  is  obtained. 

The  difference  between  the  number  of  c.c.  of  N/50 
Na2C03  used  and  the  number  of  c.c.  of  N/50  H2S04 
required  to  neutralize,  is  the  number  of  c.c.  of  N/50  sodium 
carbonate  used  up  in  precipitating  the  Ca  and  Mg.  Every 
c.c.  of  same  equals  1  c.c.  of  N/50  CaC03  =  1  mgr. 
CaC03.  Thus  we  arrive  at  the  amount  of  permanent 
hardness  in  the  quantity  of  sample  taken. 

Total. — The  sum  of  the  temporary  and  permanent 
hardnesses,  as  determined  above,  gives  the  total  hardness. 

ORGANIC  MATTER  IN  WATER. 

This  is  derived  from  vegetable  and  animal  pollution, 
and   is   estimated   in   a  variety  of  ways. 

Frankland's  Method.- — The  water  is  evaporated  to  a 
residue,  which  is  ignited  in  a  hard  combustion  tube  with 
cupric  oxide ;  the  evolved  gases  are  collected  and  measured, 
and  the  amount  of  carbon  and  nitrogen  found  in  these 
returned  as  Organic  C  and  Organic  N.  In  a  good  water 
suitable  for  domestic  use,  the  Organic  C  should  not  exceed 
0-2  part  per  100,000,  and  the  Organic  N  should  not  exceed 
0-02  part  per  100,000.  The  ratio  of  Organic  C  to 
Organic  N  furnishes  a  valuable  indication  of  the  nature 
of  the  organic  matter  present  in  unoxidized  waters.  Thus, 
unoxidized  peaty  waters  give  a  high  ratio  of  from  8  to  12 
to  20  or  even  more,  the  average  being  about  12,  and  such 
a  ratio  is  held  to  indicate  organic  matter  of  vegetable 
rather  than  of  animal  origin.    In  unpolluted  upland  surface 


WATER    ANALYSIS 


39 


water,  the  ratio  varies  from  6  to  12  ;  in  surface  water  from 
cultivated  lands,  from  4  to  10 ;  in  shallow  wells,  from  2  to 
8 ;  in  deep  wells,  and  springs,  from  2  to  6  ;  in  sea  water  it 
averages  about  17  ;  and  in  sewage  it  varies  from  1  to  3, 
averaging  about  2. 

Table. 


Unoxidized  peaty  waters 

Unpolluted  upland  surface  waters  . . 

Surface  water  from  cultivated  land 

Shallow  wells 

Deep  wells,  and  springs 

Sea  water 

Sewage 


Organic  C. 


8  to  20  ;  avge.  12 

6  to  12 

4  to  10 

2  to     8 

2  to  6 
Avge.  1-7 
1  to  3  ;  avge.  2 


Organic  N. 


In  waters  subjected  to  oxidation,  the  ratio  tends  to 
be  reduced  when  the  organic  matter  is  mainly  vegetable, 
and  the  reverse  when  it  is  animal.  Loch  Katrine 
water  (average  of  five  years)  gave  Organic  C,  0-148  part 
per  100,000,  and  Organic  N,  0-016  part  per  100,000,  and 
the  ratio  as  9-2.     This  method  is  for  trained  chemists  only. 

Wanklyn,  Chapman,  and  Hall's  Method  recognizes  that 
organic  matter  tends  to  resolve  itself  into  simpler  substances, 
and  chooses  to  estimate  the  amount  of  ammonia  present, 
free  in  solution  or  as  salts,  as  an  index  of  the  amount  of 
organic  matter  so  resolved.  Further,  the  water  is  so 
treated  subsequently  that  any  organic  matter  remaining 
undecomposed  has  its  nitrogen  split  off  as  ammonia ; 
this  is  measured  and  furnishes  an  index  of  the  amount  of 
such  organic  matter.  The  absolute  amounts  of  these  two 
ammonias  (the  first  called  "  the  free  and  saline,"  the 
second  "  the  albuminoid  ammonia  "),  and  their  relative 
amounts,  give  valuable  evidence  of  the  state  of  a  water 
with  regard  to  organic  pollution. 

Forschammer  Process,  as  modified  by  Tidy,  is  commonly 
called  Tidy's  Process,  and  consists  in  measuring  the  oxygen- 
consuming  or  absorbing  power  of  a  water,  and  inferring 
therefrom  the  amount  of  organic  matter  present.  It  has 
many  limitations,  but  under  proper  conditions  furnishes 
another  item  on  which  to  found  an  estimate  of  a  water. 


40  PUBLIC    HEALTH    CHEMISTRY 

Kjeldahl's  Process,  in  which  the  organic  nitrogen  is 
converted  into  ammonia  and  estimated  by  distillation 
along  with  the  free  and  saline  ammonia.  This  method  is 
very  useful  in  highly  polluted  waters  and  sewage  effluents, 
where  the  estimation  of  the  albuminoid  ammonia  is  tedious 
and  difficult.  It  is  much  used  to  determine  the  total 
nitrogen  in  food-stuffs,  from  which  the  total  proteins  is 
got  by  multiplying  by  a  factor  which  for  meat  foods  is 
6-25,  and  varies  for  other  foods. 

Free  and  Saline  Ammonia. — 

By  Wanklyn's  Method. — Solutions  required  :  (1)  Nessler's 
reagent.  This  is  a  saturated  solution  of  mercuric-potassic- 
iodide  in  ammonia-free  distilled  water,  the  whole  being 
rendered  strongly  alkaline  with  caustic  soda  or  potash. 
It  may  be  made  thus  :  Dissolve  35  grm.  of  potassium 
iodide  in  200  c.c.  of  ammonia-free  distilled  water  and 
12 -5  grm.  of  corrosive  sublimate  in  300  c.c.  of  ammonia- free 
distilled  water.  Add  the  iodide  solution  to  the  sublimate 
one,  when  a  yellow  to  scarlet  precipitate  is  obtained,  which 
re-dissolves  in  the  excess  of  potassium  iodide  present. 
(Mercuric  iodide  is  almost  insoluble  in  water.) 

HgCl2  +  2KI  =  Hgl2  +  2KCI. 
Hgl2    +2KI  =  HgI2.2KL 

Now  add  carefully  a  cold  saturated  solution  of  corrosive 
sublimate,  stirring  all  the  time,  until  a  slight  red  precipitate 
remains  permanent.  In  this  way  excess  of  potassium 
iodide,  above  that  required  to  keep  the  mercuric  iodide 
in  solution,  is  used  up.  120  grm.  of  caustic  soda  in  stick 
are  now  added  to  the  mixture  and  allowed  to  dissolve 
and  cool.  If  the  red  precipitate  has  disappeared,  add 
again  a  little  of  the  saturated  solution  of  corrosive  sublimate, 
until  a  slight  permanent  red  precipitate  appears.  Make 
up  bulk  to  1  litre  with  ammonia-free  distilled  water.  The 
solution  is  now  ready  for  use. 

Nessler's  solution  gives  a  yellowish  tinge  with  the 
faintest  trace  of  ammonia,  and  if  much  ammonia  is  present 
a  yellow-brown  precipitate  forms  of  di-mercuric-ammonium- 
iodide  : — 

NH3  +  2HgI2  =  NHg2I  +  3HL 


WATER    ANALYSIS  41 

(2)  Standard  solution  of  ammonium  chloride,  such  that 
1  c.c.  =  o-oi  mgr.  NH3.  Ammonium  chloride,  NH4C1, 
has  a  molecular  weight  of  53-5,  of  which  17  parts  are  due 
to  ammonia  NH  3.  Since  the  standard  solution  is  1  c.c.  = 
o-oi  mgr.  of  ammonia,  1  litre  will  contain  o-oi  grm.  of 
ammonia. 

17  :  o-oi  :  :  53-5  :  x  =  0-03147  grm.  of  NH4C1  will  yield 
o-oi  grm.  of  ammonia.  Hence,  dissolve  0-03147  grm.  of 
ammonium  chloride  in  1  litre  of  ammonia-free  distilled 
water,  and  1  c.c.  will  contain  o-oi  mgr.  of  ammonia. 

Process. — Take  a  retort  or  boiling-flask  of  about  700  c.c. 
capacity,  cleanse  it  well  and  rinse  it  out  with  ammonia- 
free  distilled  water.  Now  put  into  it  200  c.c.  of  ammonia- 
free  distilled  water,  connect  to  a  condenser,  start  the  water 
flow  in  latter,  and  distil  over  100  c.c.  to  rid  the  apparatus 
of  any  traces  of  ammonia.  Test  the  distillate  with  Nessler's 
solution,  and  if  ammonia  is  found  in  the  last  portions, 
repeat  the  distillation.  If  not,  cool  flask,  wash  out  with 
ammonia-free  water,  and  proceed. 

Introduce  into  flask  500  c.c.  of  sample  water  and  render 
this  alkaline  by  the  addition  of  some  recently-heated 
sodium  carbonate.  Connect  to  a  condenser,  start  the  water 
supply  for  the  latter,  and  place  a  clean  50  c.c.  Nessler  glass 
at  the  end  of  the  condenser  to  catch  the  distillate.  Make 
sure  that  all  parts  of  the  apparatus  are  properly  connected 
and  adjusted.  Now  apply  the  flame  of  a  Bunsen  burner 
to  the  flask,  which  may  be  protected  by  a  piece  of  gauze. 
Heat  gently  at  first,  but  once  the  parts  have  got  heated, 
increase  the  flame.  Try  to  distil  over  at  the  rate  of  50  c.c. 
every  fifteen  minutes.  When  the  first  Nessler  glass  is 
filled  to  the  50  c.c.  mark,  remove  it  and  put  another  clean 
one  in  its  place,  and  so  on.  Have  a  stock  of  six  ready  for 
the  purpose. 

The  first  50  c.c.  of  distillate  is  then  tested  by  adding  to 
it  2  c.c.  of  Nessler  solution  and  mixing.  Place  the  glass 
on  a  white  slab,  or  on  the  glass  shelf  of  a  Nessler  stand, 
and  on  looking  down  through  the  liquid,  the  amount  of 
coloration  produced,  or  its  absence,  is  easily  made  out. 
With  experience  the  depth  of  colour  will  suggest  how 
much  standard  solution  will  be  required  to  match  it. 
The  next  step  is  to  put  up  three  trial  glasses  for  comparison. 


42  PUBLIC    HEALTH    CHEMISTRY 

Take  three  50  c.c.  Nessler  glasses,  and  from  a  burette  add 
to  the  first  1  c.c.,  to  the  second  2  c.c,  and  to  the  third 
3  c.c.  of  standard  solution  of  ammonium  chloride ;  1  c.c. 
=  o-oi  mgr.  NH  3.  Fill  all  three  up  to  the  50  c.c.  mark 
with  ammonia-free  distilled  water,  and  then  add  to  each 
2  c.c.  of  Nessler  solution,  and  mix.  Put  these  glasses, 
distinctively  marked,  on  the  slab  or  shelf,  and  compare 
the  first  50  c.c.  of  distillate  with  them  as  to  depth  of 
coloration.  If  the  distillate  matches  any  one  of  them, 
the  result  is  attained.  If  it  does  not  match  any  of  them, 
it  may  be  intermediate  between  any  two  of  them,  or  be 
darker  than  the  3  c.c.  or  lighter  than  the  1  c.c.  glass.  In 
any  case,  fresh  trial  glasses  should  be  put  up  for,  as.  the 
case  may  be,  -5  c.c,  1-5  c.c,  2-5  c.c,  4  c.c,  5  c.c,  6  c.c, 
of  standard  solution.  If  the  distillate  is  darker  than  the 
coloration  given  by  6  c.c.  of  standard  solution,  it  is  better 
to  dilute  it  with  ammonia-free  distilled  water,  and  then  to 
proceed  to  match.  The  same  procedure  is  carried  out 
with  the  second  50  c.c.  of  distillate  and  succeeding  lots. 
The  distillation  is  stopped  when  no  coloration  is  given 
with  2  c.c  of  Nessler,  or  at  least  less  than  will  match  0-5  c.c 
of  standard  solution.  This  usually  happens  with  the 
fourth  lot  of  50  c.c.  The  sum  of  the  amounts  of  ammonia 
found  in  each  lot  of  distillate,  is  the  total  free  and  saline 
ammonia  present  in  500  c.c.  of  sample  water.  This  is 
reduced  to  the  amount  in  100  c.c,  and  thereafter  expressed 
as  parts  of  ammonia  per  100,000.  The  distilling  over  in 
separate  lots  is  the  mode  recommended  by  the  Society  of 
Public  Analysts,  but  Wanklyn  recommends  that  only 
50  c.c.  be  distilled,  and  that  the  amount  found  in  it  be 
increased  by  one-third,  on  the  ground  that  in  his  experience 
three-fourths  of  the  ammonia  comes  over  in  the  first  lot 
of  50  c.c. 

Example. — 
First       50  c.c.  of  dist.  matched  5.0  c.c.  std.  sol.  of  NH4C1. 
Second    50  c.c       do.         do.       1-5  c.c.       do.  do. 

Third      50  c.c.       do.         do.       0-5  c.c       do.  do. 

Fourth    50  c.c.       do.         do.       nil 

7-0  c.c      do.  do. 


WATER    ANALYSIS  43 

That  is,  the  500  c.c.  of  sample  water  yielded  ammonia 
sufficient  to  match  7  c.c.  of  standard  solution  of  ammo- 
nium chloride.  (1  c.c.  =  o-oi  mgr.  NH3),  hence  7  c.c. 
standard  solution  equals  7  X  o-oi  —  0-07  mgr.  NH  3,  and 
there  is — 

0-07    mgr.  of  free  and  saline  NH3  in  500  c.c.  of  sample. 
or  0-014  mgr.  of       do.       do.       do.    in  100  c.c. 
or         do.  in  100  grm. 

or  do.  in  100,000  mgr. 

or  0-014  Part  of        do.       do.       do.    in  100,000  parts. 

Note. — In  Nesslerizing,  always  add  the  AmCl  solution 
first  to  the  trial  glasses,  and  the  Nessler  later.  If  the  order 
is  reversed,  a  turbidity  is  produced  which  prevents  accurate 
comparisons. 

Likewise  always  use  distilled  water,  ammonia-free. 
Pure  natural  water,  ammonia-free,  will  not  do,  as  it  appears 
muddy  when  compared  with  distilled  water. 

The  residue  in  the  flask  is  used  to  determine  the 
"  albuminoid  ammonia,"  as  now  described. 

Albuminoid  Ammonia — is  determined  by  breaking  up 
the  nitrogenous  organic  matter  in  the  water  sample  by  the 
action  of  an  alkaline  solution  of  potassium  permanganate, 
the  nitrogen  being  converted  into  ammonia,  which  is  distilled 
off  and  estimated  as  described  for  free  and  saline  ammonia. 
All  the  nitrogenous  organic  matter  is  not  so  decomposed, 
but  the  proportion  of  it  which  does  so  is  sufficiently  uniform 
to  form  a  basis  for  deductions.  The  albuminoid  ammonia 
is  approximately '  one-tenth  of  the  nitrogenous  organic 
matter  in  the  water.  Solution  required  is  "  alkaline 
permanganate,"  made  by  dissolving  8  grm.  of  potassium 
permanganate  and  200  grm.  of  caustic  potash  in  1100  c.c. 
of  distilled  water,  and  boiling  down  the  bulk  to  1000  c.c. 
(1  litre). 

The  amount  of  alkaline  permanganate  used  should  be 
about  one-tenth  of  the  bulk  of  sample  taken.  It  should 
be  mixed  with  three  volumes  of  water,  and  boiled  down  to 
three  volumes.  That  is,  in  present  case,  take  50  c.c.  of 
alkaline  permanganate,  add  to  them  150  c.c.  of  water,  and 
boil  down  to  150  c.c,  which  are  added  to  the  residue  in 


44  PUBLIC    HEALTH    CHEMISTRY 

flask.  This  gets  rid  of  any  free  or  saline  or  albuminoid 
ammonia  in  this  added  fluid. 

Process. — Take  the  residue  from  the  estimation  of 
free  and  saline  ammonia,  and  keeping  it  still  in  the  flask, 
add  to  it  50  c.c.  of  freshly-boiled  permanganate  solution, 
and  about  100  c.c.  of  ammonia-free  distilled  water,  to 
increase  the  bulk.  Some  fragments  of  pumice  stone,  which 
have  been  heated  to  redness  in  a  Bunsen  flame  and  cooled, 
are  also  added.  The  apparatus  is  now  fixed  together 
and  distillation  resumed,  the  distillate  being  collected  as 
before  in  50  c.c.  Nessler  glasses.  The  determination  of 
the  amount  of  ammonia  is  made  in  precisely  the  same 
way  as  for  free  and  saline  ammonia.  The  number  of  lots 
of  50  c.c.  to  be  collected  cannot  be  approximately  stated, 
as  the  splitting  up  of  the  organic  matter  occurs  irregularly, 
and  in  this  way  more  ammonia  may  be  found  in  the  second 
or  third  lot  than  in  the  first.  The  process  should  be 
continued  until  no  reaction  with  Nessler  is  got.  In  some 
cases  it  may  be  necessary  to  stop  the  process  and  allow 
the  apparatus  to  cool,  then  add  more  distilled  water, 
and  then  resume  the  distillation.  The  amount  of  ammonia 
found  is  of  course  derived  from  the  original  500  c.c,  and 
must  be  calculated  accordingly. 

Free  and  saline  ammonia  represents  the  ammonia 
combined  with  carbonic,  nitric,  or  other  acids,  and  also 
what  may  be  derived  from  urea,  or  other  easily  decom- 
posable substances,  if  present.  The  limit  in  pure  waters 
is  0-002  mgr.  per  100  c.c,  and  in  a  usable  water  it  should 
not  exceed  0*005  nigr.  per  100  c.c. 

Albuminoid  ammonia  in  drinking  waters  of  good 
quality  should  not  exceed  o-oi  parts  per  100,000.  Much 
albuminoid  ammonia  with  a  small  amount  of  free  ammonia 
usually  indicates  vegetable  contamination,  particularly  if 
the  chlorides  and  nitrates  are  low.  Peaty  waters  yield 
large  quantities  of  albuminoid  ammonia,  which  is  slowly 
evolved  ;  whereas  badly  polluted  waters  as  a  rule  yield 
their  high  proportion  more  rapidly. 

Oxygen  Absorption  or  Consuming  Power. — Tidy's 
process  is  based  on  the  fact  that  much  of  the  organic 
matter  in  a  water  is  capable  of  oxidation,  and  especially 
by  permanganate  in  acid  solution.     Unfortunately,  different 


WATER    ANALYSIS  45 

substances  reduce  different  proportions  of  permanganate, 
and  slight  variations  in  temperature  and  acidity  influence 
the  readiness  of  the  permanganate  to  part  with  its  oxygen 
to  an  appreciable  extent.  Nevertheless,  the  process  yields 
results  which,  taken  in  conjunction  with  other  analytical 
facts,  aid  materially  in  forming  an  opinion  of  a  water 
sample. 

Reagents  required  :  (a)  Standard  solution  of  potassium 
permanganate  : — 

2K  2Mn  20  8+6H  2SO  4=2K  2SO  4-f  4MnSO  4+6H  20+50  2 

2  x  316  5  x  32  ; 

that  is,  632  parts  of  potassium  permanganate  liberate  160 
parts  of  oxygen,  or  1  part  of  O  will  be  set  free  by  3-95  parts 
of  permanganate.  Hence,  if  3-95  grm.  of  the  latter  be 
dissolved  in  1  litre  of  aq.  dest.,  then  1  c.c.  =  1  mgr.  O. 
The  solution  is  usually  diluted  ten  times  in  use,  so  that 
10  c.c.  =*  1  mgr.  O.  (b)  KI  solution,  10  per  cent  in  aq. 
dest.  (c)  Starch  solution,  1  grm.  per  half  litre,  freshly 
boiled  and  filtered,  (d)  Sodium  thiosulphate  solution, 
1  grm.  to  the  litre  of  distilled  water,  (e)  Sulphuric  acid, 
25  per  cent  in  aq.  dest. 

Process. — Take  two  stoppered  flasks  or  bottles  of  at 
least  300  c.c.  capacity,  and  into  one  put  250  c.c.  of  sample 
water,  and  into  the  other  put  250  c.c.  of  distilled  water. 
To  each  add  10  c.c.  of  the  25  per  cent  sulphuric  acid,  and 
place  them  both  on  a  water-bath  at  8o°  F.  or  260  C.  When 
the  required  temperature  is  reached,  10  c.c.  of  the  perman- 
ganate solution  are  added  to  each  lot.  A  pink  colour  will 
result.  Maintain  the  temperature,  and  observe  carefully 
whether  the  pink  colour  is  discharged.  If  so,  then  another 
10  c.c.  of  the  permanganate  solution  is  added  to  the  sample 
and  the  control,  and  more  if  necessary  to  keep  them 
markedly  pink.  Further  addition  of  sulphuric  acid  is  not 
needed.  At  the  end  of  a  specified  time,  which  may  be 
fifteen  minutes,  half  an  hour,  one  hour,  two  hours,  three 
hours,  or  four  hours,  or  any  combination  of  these  (the 
commonest  being  fifteen  minutes  and  four  hours),  the 
oxidizing  process  is  stopped  by  the  addition  of  1  c.c.  of  the 
KI  solution,  when  the  unused  permanganate  reacts  thus,, 
through  its  loosely  held  oxygen  ;  5O  2  +  20KI  +  10H  20  =. 


46  PUBLIC    HEALTH    CHEMISTRY 

20KOH  +  10I 2.  The  liquid  turns  a  yellow  colour  from 
the  iodine  set  free.  The  quantity  of  iodine  liberated  is 
strictly  proportional  to  the  amount  of  unused  perman- 
ganate. It  remains,  therefore,  to  estimate  the  amount  of 
iodine  set  free,  which  measures  the  amount  of  oxygen 
unused,  and  this  deducted  from  the  amount  known  to 
have  been  added,  gives  the  amount  absorbed.  This  is 
done  by  titrating  the  yellow  solution  with  the  thiosulphate 
solution  until  the  yellow  colour  is  nearly  gone,  and  then 
adding  1  c.c.  of  starch  solution  to  give  a  more  distinct  end 
reaction.  The  titration  is  finished  when  the  blue  is  just 
gone  : — 

1 2  +  2Na  2S  20  3  =  2NaI  -f-  Na  2S  40  6. 

Both  the  sample  and  the  control  are  treated  thus.  In  the 
control  presumably  no  oxidation  takes  place,  so  that  the 
number  of  c.c.  of  thiosulphate  solution  required  for  it,  is  a 
measure  of  the  iodine  liberated  by  all  the  oxygen  free  to 
cause  oxidation.  The  amount  of  thiosulphate  solution  used 
for  the  sample  measures  the  unused  oxygen,  and  the  differ- 
ence between  these  two  numbers  gives  the  proportion  of 
oxygen  used  up.  For  example,  say  that  10  c.c.  only  of  per- 
manganate were  required  to  be  used,  and  that  after  adding 
KI  solution  the  control  took  40  c.c.  of  thiosulphate  solution 
to  decolorize,  and  the  sample  took  30  c.c.  ;  then  40  c.c. 
of  thiosulphate  measure  1  mgr.  of  oxygen,  and  40  c.c. 
—  30  c.c.  =  10  c.c.  measure  10/40  =  0-25  mgr.  O,  and 
this  is  the  quantity  absorbed  by  the  250  c.c.  of  sample 
taken.  This  multiplied  by  0-4  gives  o-i  mgr.  O  absorbed 
per  100  c.c,  or  o-i  part  per  100,000. 

Waters  of  great  organic  purity  will  not  consume  more 
than  0-05  part  of  oxygen  per  100,000  in  fifteen  minutes 
at  8o°  F.,  and  if  the  amount  absorbed  exceeds  o-i  part  in 
fifteen  minutes,  the  sample  may  be  considered  of  doubtful 
purity.  After  four  hours'  exposure,  an  absorption  of  more 
than  0-3  part  must  be  regarded  with  suspicion. 

Ferrous  salts,  nitrites,  and  sulphuretted  hydrogen,  if 
present,  vitiate  the  test. 

Kjeldahl's  Process  for  the  determination  of  organic 
nitrogen  is  performed  only  in  very  polluted  waters.  The 
process  is  described  under  Sewage  and  Sewage  Effluents. 


WATER    ANALYSIS  47 

NITRITES     AND     NITRATES     IN     WATER. 

Ammonia  present  in  water,  derived  either  from  the 
decomposition  of  organic  matter  or  by  synthesis  from  urea, 
tends  in  its  passage  through  the  soil  to  become  oxidized, 
first  into  nitrites,  then  into  nitrates.  Nitrates,  however, 
may  be  present  in  water  which  has  dissolved  it  out  of  strata 
through  which  it  has  passed.  Sometimes  these  nitrates 
become  reduced,  first  to  nitrites,  then  to  ammonia,  and  this 
has  been  specially  observed  as  due  to  iron  salts  in  the  ferrous 
state.  In  the  London  basin,  the  deep-well  waters  from  the 
"  greensands  "  strata  have  been  noted  as  yielding  ammonia 
thus  derived.  Waters  polluted  with  vegetable  matter  yield 
little  nitrites  and  nitrates  relatively,  as  plant  life  removes 
these,  and  vegetable  matter  contains  little  nitrogen. 

Nitrites. — 

Qualitative   Tests. 

i.  Starch  Iodine  Test. — Take  50  c.c.  of  sample  water  in  a 
Nessler  glass  and  50  c.c.  distilled  water  in  another.  To 
each  add  a  few  drops  of  KI  solution  and  a  few  drops  of 
freshly-made  starch  solution.  Now  add  a  few  drops  of 
dilute  sulphuric  acid  to  each  tube.  The  presence  of 
nitrites  is  indicated  by  an  immediate  blue  colour. 

2.  Naphthylamine  Test. — Take  two  Nessler  glasses  as  above 
and  acidulate  with  acetic  acid.  Add  to  each  a  few  drops  of 
naphthylamine  solution  in  sulphanilic  acid.  With  nitrites 
a  beautiful  pink  colour  develops  in  two  to  three  minutes. 

Quantitative   Tests. 

Griess's  Test. — Solutions  required  : — 

a.  Metaphenylene-diamine  solution,  5  grm.  per  litre, 
slightly  acidulated  with  sulphuric  acid,  decolorized  by 
boiling  with  pure  animal  charcoal. 

b.  Sulphuric  acid,  one  part  of  strong  acid  to  two  parts 
of  aq.  dest. 

c.  Standard  nitrite  solution.  This  is  made  from  silver 
nitrite  because  it  is  the  most  stable  salt.  AgNO  2+KCl  = 
AgCl  +  KNO  2.  154  parts  of  silver  nitrite,  when  treated 
with  KG,  give  rise  to  85  parts  of  KNO  2,  or  46  parts  of 
nitrous  acid  as  represented  by  N02,  or  308  parts  are 
equivalent  to  76  parts  of  N  20  3.     Hence,  if  308  /y6  =  4-06 


48  PUBLIC    HEALTH    CHEMISTRY 

grm.  silver  nitrite  are  dissolved  in  boiling  distilled  water, 
and  precipitated  by  slight  excess  of  KC1,  I  grm.  of  nitrous 
acid  as  N203  is  left  in  solution  in  combination  with 
potassium.  The  bulk  is  made  up  to  I  litre,  the  precipitate 
allowed  to  settle,  and  10  c.c.  are  taken  and  diluted  to  I  litre, 
then  i  c.c.  =  o-oi  mgr.  N203  (equal  to  i  per  100,000). 
Standard  nitrite  solution  is  also  made  so  that  1  c.c. 
=  o-oi  mgr.  N,  and  also  of  millinormal  strength,  and 
then  1  c.c.  N/1000  =  0-046  mgr.  N02. 

Process. — To  100  c.c.  of  sample  in  a  Nessler  glass,  1  c.c. 
of  the  dilute  sulphuric  acid  and  1  c.c.  of  the  metaphenylene- 
diamine  solution  are  added  as  a  preliminary  test.  If  an 
orange  colour  is  immediately  produced,  the  tint  will  prove 
too  deep  for  comparison.  In  such  a  case  50  c.c.  should 
be  tried,  and  if  found  suitable  such  an  amount  diluted  to 
100  c.c.  with  aq.  dest.  is  to  be  used  in  the  real  test,  which 
is  done  thus  : — Having  decided  the  amount  of  the  sample 
to  be  used,  it  is  taken  in  a  100  c.c.  Nessler  glass  and  made 
up  to  100  c.c.  if  required.  Three  other  Nessler  glasses  are 
taken,  and  1  c.c,  2  c.c,  and  3  c.c.  of  standard  nitrite 
solution  added  to  each  respectively,  and  the  bulk  is  made 
up  to  100  c.c  in  each  case  with  aq.  dest.  To  all  the  glasses 
is  added  1  c.c.  of  each  of  the  reagents,  namely  M-P-D 
and  H2S04,  and  this  is  done  as  quickly  as  possible,  so 
that  the  colours  in  the  glasses  may  develop  from  as  nearly 
as  possible  the  same  time.  The  glasses  are  set  aside  for 
fifteen  to  twenty  minutes  and  are  then  compared  in  the 
manner  known  as  "  Nesslerizing."  If  the  sample  matches 
one  of  the  standards,  then  the  amount  in  it  is  known. 
If  not,  fresh  trial  glasses  are  put  up,  the  amount  of  standard 
being  gauged  from  the  preceding  experiment. 

The  colour  produced  is  Bismarck  brown  or  triamido-azo- 
benzol.  Griess's  test  is  a  very  accurate  one  but  requires  that 
the  water  and  the  reagent  should  be  colourless  or  be  decolor- 
ized.   The  reagent  may  be  bleached  by  pure  animal  charcoal. 

Ilosvay's  N aphthylamine  Test. — 

a.  Solution  of  sulphanilic  acid  0-5  grm.  in  150  c.c  of 
diluted  acetic  acid  (specific  gravity  1-04). 

b.  Solution  of  naphthylamine  made  by  dissolving  o-i 
grm.  in  20  c.c  of  aq.  dest.,  filtering,  and  adding  180  c.c 
diluted  acetic 


WATER    ANALYSIS  49 

Process. — Take  ioo  c.c.  of  sample  in  a  Nessler  glass,  and 
in  another  the  same  quantity  of  aq.  dest.  and  I  c.c.  standard 
nitrite  solution.  To  each  add  2  c.c.  of  each  of  the  above 
solutions,  a  and  b.  Set  aside  for  five  minutes  and  then 
compare  tints.  If  not  equal  in  tint,  abstract  some  fluid 
from  the  darker  by  pipette  and  make  up  the  bulk  with 
aq.  dest.  If  the  colours  still  do  not  match,  more  fluid  is 
removed,  and  bulk  made  up  as  before.  Suppose  sample 
is  darker,  and  that  40  c.c.  are  removed,  and  bulk  made  up  ; 
and  that  again,  30  c.c.  are  removed,  when  finally  tints 
match.  Then  we  get  : — 100  x  60/100  x  70/100  =  42  c.c. 
of  the  original  100  c.c.  match  1  c.c.  of  standard  nitrite 
solution,  say  1  c.c.  =  o-oi  mgr.  N  :  then  42  c.c.  of  sample 
contain  o-oi  mgr.  N,  and  therefore  100  c.c.  will  contain 
o-oi  x  100  ~-  42  =  0-023  mgr-  N,  or  0-023  Part  of  N 
as  nitrite  per  100,000  parts. 

The  nitrites  first  act  on  the  sulphanilic  acid  and  form 
a  new  compound  which  reacts  with  the  naphthylamine 
and  forms  the  substance  which  gives  the  pink  colour  to 
the  liquid. 

A  water  containing  nitrites  is  not  safe  for  domestic  use, 
and  should  be  rejected  on  that  evidence  alone,  unless 
unexceptionable  in  all  other  respects. 

Nitrates. — 

Qualitative   Tests. 

1.  Brucine  Test. — Take  5  c.c.  of  sample  and  add  5  c.c.  of 
brucine  solution  (1  in  1000),  then  mix,  and  pour  carefully 
down  the  side  of  the  test  tube  some  pure,  strong  sulphuric 
acid,  free  from  nitrates,  when  a  positive  result  is  denoted 
by  the  appearance  of  a  pink  ring  at  the  junction  of  the 
liquids  on  gentle  shaking.  The  test  is  also  performed  by 
evaporating  10  c.c.  of  sample  to  dryness  in  a  clean  porcelain 
basin,  then  adding  a  crystal  of  brucine,  and  then  allowing 
one  drop  of  pure  sulphuric  to  run  down  the  side  of  the 
dish,  over  the  solids  ;  when  in  the  presence  of  nitrates 
a  pink  is  obtained.  Detects  0-7  part  per  100,000. 
Unreliable  in  the  presence  of  nitrites,  which  should  be 
first  destroyed  by  addition  of  urea  and  sulphuric  acid  to 
sample  ;  allow  to  stand  aside  for  an  hour,  when  test  can 
be  applied  as  before. 

4 


50  PUBLIC    HEALTH    CHEMISTRY 

2.  Diphenylamine  Test  (C6H5)2NH. — Take  5  c.c.  of 
sample,  add  as  much  diphenylamine  solution,  mix,  and 
run  down  pure  strong  sulphuric,  when  a  blue  colour  forms 
at  junction  of  liquids  in  presence  of  nitrates. 

Quantitative   Tests. 

1.  Phenol-sulphonic  Acid. — This  reagent  is  made  by 
adding  6  grm.  of  pure  carbolic  acid  to  3  c.c.  of  aq.  dest. 
and  then  adding  mixture  to  37  c.c.  of  pure  sulphuric  acid. 

A  standard  solution  of  potassium  nitrate  is  required, 
0-072  grm.  to  1  litre,  and  then  1  c.c.  =  o-oi  mgr.  N  as 
nitrates.  This  contains  1  part  N  in  100,000  parts  of 
standard. 

Process. — To  two  porcelain  dishes  are  added  respectively 
10  c.c.  of  the  sample  and  10  c.c.  of  the  standard.  These 
are  placed  on  the  water-bath  until  their  contents  are  just 
evaporated  to  dryness.  To  each  of  the  residues  add  1  c.c. 
of  phenol-sulphonic  acid,  and  mix  well  with  a  glass  rod  (if 
a  large  amount  of  nitrates  is  present  the  liquid  will  turn 
red).  Set  aside  for  fifteen  minutes,  and  then  wash  out 
each  dish  successively  into  two  clean  100  c.c.  Nessler 
glasses  with  25  per  cent  ammonia  solution  in  distilled 
water.  Add  more  ammonia  until  effervescence  ceases, 
and  make  up  to  mark  with  aq.  dest.  The  nitrates  present 
convert  the  phenol-sulphonic  acid  into  picric  acid,  with 
which  the  ammonia  forms  a  picrate  having  a  yellow  colour, 
and  the  amount  of  this  is  proportional  to  the  amount  of 
nitrates  present.  The  two  glasses  are  now  compared  as 
to  tints,  and  the  darker  one  is  diluted  as  before  described 
under  Ilosvay's  test  for  nitrites.  If  the  water  is  very 
pure,  a  larger  amount  of  sample  should  be  evaporated 
down,  say  20  c.c,  50  c.c,  or  100  c.c,  and  a  smaller  quantity 
of  standard,  say  5  c.c.  If  rich  in  nitrates,  then  less  should 
be  taken  of  the  sample,  say  5  c.c  or  1  c.c 

Aluminium  Process. — If  aluminium  foil  be  added  to  a 
strongly  alkaline  water,  decomposition  of  the  water  ensues 
with  the  evolution  of  hydrogen,  which  in  the  presence  of 
nitrites  or  nitrates  reduces  these,  converting  their  contained 
nitrogen  into  ammonia.     Thus  : — 

4AI  +  4NaOH  +  4H20  =  2Al2Na204  +  6H2  :  and 
3KN03  +  I2H2  =  3KOH  +  6H20  +  3NH3. 


WATER    ANALYSIS  51 

Required  :  (i)  Thin  aluminium  foil ;  (2)  10  per  cent 
solution  NaOH. 

Process. — Take  100  c.c.  of  the  water  sample  and  100  c.c. 
of  the  NaOH  solution  in  a  300  c.c.  boiling-flask.  Add  a 
piece  of  aluminium  foil  about  1-5  inch  square.  Cover  but 
do  not  cork.  Set  aside  for  six  hours  at  least.  Then 
connect  the  flask  to  a  condenser  and  distil  over  the  ammonia 
into  50  c.c.  Nessler  glasses,  collecting  three  lots.  The 
amount  of  ammonia  is  determined  by  comparison  of  the 
coloration  developed  in  these  glasses  by  adding  to  each 
2  c.c.  Nessler's  solution,  and  that  developed  in  glasses 
containing  50  c.c.  ammonia- free  distilled  water,  plus  1  c.c, 

2  c.c,  and  3  c.c  respectively  of  standard  NH4C1  (1  c.c. 
=  o-oi  mgr.  NH3),  and  similarly  treated.  The  amount 
estimated  by  this  method  includes  ammonia  present  in 
the  sample,  ammonia  derived  from  nitrites,  and  ammonia 
derived  from  nitrates.  The  two  former  are  separately 
estimated  and  deducted,  and  the  remainder  is  the  amount 
derived  from  nitrates,  and  is  readily  converted  back  into 
terms  of  NO  3  or  of  N. 

Example. — 100  c.c  gave  ammonia  equal  to  40  c.c  of 
standard  AmCl  =  40  x  o-oi  =  0-4  mgr.  NH3.  But  the 
water  contained  0-006  mgr.  of  free  and  saline  ammonia 
per  100  c.c,  and  0-042  mgr.  N  as  nitrites  per  100  c.c  = 
0-042  x  17  -5-  14  =  0'05iNH3  per  100  c.c.  Hence,  0-4  — 
(0-006  4-  0*051)  =  0-343  mgr.  NH  3  due  to  nitrates  =  0-282 
mgr.  N  as  nitrates  per  100  c.c.  or  100,000  mgr. 

Copper-Zinc  Couple  Method. — This  method  is  similar  in 
principle  to  the  above.     A  bright  piece  of  thin  zinc  foil, 

3  in.  X  2  in.,  is  cleansed  with  dilute  sulphuric  acid.  It  is 
then  rolled  into  a  coil,  so  that  it  may  fit  into  a  200  c.c. 
wide-mouthed  bottle.  Now  immerse  the  coil  for  three 
minutes  in  a  3  per  cent  solution  of  copper  sulphate.  The 
zinc  becomes  coated  with  a  black  deposit  of  metallic 
copper.  Remove  the  coil  carefully,  wash  in  ammonia-free 
distilled  water,  wash  in  sample  water,  and  then  immerse 
in  no  c.c  of  sample  water  contained  in  a  wide-mouthed 
bottle.  Stopper  tightly,  place  in  a  cool  dark  place  for 
twenty-four  hours.  The  "  Copper-zinc  couple "  acts 
electrically  on  the  sample,  changing  any  nitrates  present 


52  PUBLIC    HEALTH    CHEMISTRY 

to  nitrites,  and  then  to  ammonia.  It  thus  acts  also  on 
any  nitrites  originally  present,  so  that  the  process  estimates 
nitrates  and  nitrites.  The  reaction  is  finished  when  no 
free  nitrites  are  found  in  the  solution.  This  is  determined 
by  removing  10  c.c.  and  testing  by  Griess's  test.  If 
nitrites  are  found  to  be  present,  more  time  must  be  given. 
If  they  are  absent,  the  remainder  of  the  sample  water  is 
poured  into  a  700  c.c.  boiling-flask,  and  the  bottle  washed 
out  repeatedly  with  ammonia-free  distilled  water,  the 
washings  being  added  to  the  flask,  and  more  water  added 
to  bring  up  the  bulk  to  about  500  c.c.  The  water  is  then 
distilled  as  in  the  estimation  of  free  and  saline  ammonia, 
and  the  amount  of  ammonia  determined.  This  is  restated 
as  nitrogen  by  multiplying  by  14/17  (N:NH3).  If  the 
sample  was  found  to  contain  any  free  ammonia,  the  amount 
of  this  would  require  to  be  deducted  before  assigning  the 
amount  found  by  this  process  to  nitrates  and  nitrites. 

Indigo  Method. — Another  method,  which  is  a  rapid  and 
convenient  one,  but  subject  to  great  irregularity,  is  the 
indigo  method.  20  c.c.  of  sample  are  taken  in  a  beaker, 
and  20  c.c.  of  pure  strong  sulphuric  acid  are  added.  From 
a  burette  allow  standard  indigo  solution  to  run  into  the 
hot  mixture,  until  the  colour  of  the  indigo  ceases  to  be 
discharged,  and  a  faint  greenish  tinge  becomes  permanent. 
The  estimation  should  be  repeated,  adding  half  a  c.c.  of 
indigo  less  to  20  c.c.  of  the  sample,  and  then  the  sulphuric. 
When  the  colour  is  discharged,  the  indigo  is  run  in  drop 
by  drop  until  colour  is  again  permanent.  The  indigo 
solution  is  standardized  against  standard  nitrate  solution 
similarly  treated.  The  strong  sulphuric  liberates  free 
nitric  acid,  which  in  the  hot  liquid  oxidizes  the  indigo  to 
isatin,  which  is  colourless.  Owing  to  the  heat  evolved, 
the  titration  is  best  done  with  the  beaker  resting  on  an 
asbestos  mat. 

The  method  is  unreliable  in  the  presence  of  organic 
matter,  the  results  being  too  small.  It  also  requires  that 
all  the  procedures  should  be  carried  out  exactly  alike  for 
the  titration  of  the  sample  and  the  standardizing  of  the 
indigo  solution.  Otherwise  it  is  a  very  simple,  rapid,  and 
delicate  method. 

No  water  used  for  drinking  purposes  should  contain 


WATER    ANALYSIS  53 

more  than  0-35  part  of  nitrogen  as  nitrates  per  100,000 
parts,  unless  there  is  some  satisfactory  explanation.  This 
amount  equals  about  one  grain  per  gallon  when  expressed 
as  N205,  or  1-5  parts  per  100,000  when  expressed  as  N03. 

ICE. 

Ice  is  frozen  water,  and  it  is  not  usually  purer  in 
content  than  the  water  from  which  it  is  derived.  What- 
ever may  be  frozen  out  of  the  water  is  usually  mineral 
matter,  such  as  salt ;  suspended  matter  is  likely  to  be 
enclosed.  Microbic  content,  when  composed  of  the  com- 
mon sewage  organisms,  is  little  affected  by  the  temperature 
of  freezing,  for  the  most  part  only  being  rendered  torpid. 
As  far  as  possible,  therefore,  ice  should  only  be  used  when 
made  from  pure  water,  and  by  a  process  in  which  it  is  not 
subject  to  risk  of  serious  contamination.  The  analysis  of 
ice  proceeds  on  the  same  methods  as  for  water,  the  ice 
being  first  melted. 

MINERAL     WATERS     AND     AERATED     WATERS. 

These  are  examined  on  the  same  principles.  In  the 
case  of  artificial  waters,  the  spring  or  supply  from  which 
they  are  made  should  also  be  examined.  In  such  also  a 
search  should  be  made  for  poisonous  metals,  such  as  lead 
and  antimony,  iron,  copper,  zinc,  and  even  arsenic. 

In  natural  mineral  waters  the  same  careful  examination 
should  be  made.  In  these  the  mineral  content  is  often 
considerable,  and  a  thorough  analysis  of  the  different 
metals  present  is  very  important.  The  temperature  and 
the  amount  of  carbonic  acid  gas  are  also  noted.  Nowadays 
the  presence  of  metals  of  the  radium  group  has  acquired 
a  new  significance,  and  their  occurrence  is  specially  noted. 

INTERPRETATION     OF     THE     RESULTS     OF     A 
WATER     ANALYSIS. 

This  must  not  be  based  on  any  one  item,  but  on  a 
careful  consideration  of  the  following  points  : 

1.  Local  inspection  for  any  source  of  possible  pollution. 

2.  Bacteriological  examination  made  as  soon  after  collec- 
tion as  possible. 


54  PUBLIC    HEALTH    CHEMISTRY 

3.  Chemical  analysis  made  as  soon  after  collection  as 
possible. 

It  is  rare  for  a  sample  of  water  to  yield  results  under  the 
second  and  third  headings,  where  careful  local  inspection 
has  failed  to  suggest  danger  of  pollution.  It  should,  there- 
fore, be  thoroughly  carried  out. 

Bacteriological  examination  absolutely  condemns  a  water 
for  domestic  use  when  pathogenic  organisms  are  found  in 
it.  Unfortunately  the  detection  of  these  is  not  always 
an  easy  matter,  and  so  their  presence  or  absence  is 
inferred  from  the  abundance  or  scarcity  of  associated 
forms  which  are  more  readily  found  and  identified.  The 
result  of  this  is  that  the  bacteriological  examination  mostly 
furnishes  evidence  confirmatory  to  that  derived  from 
other  sources. 

Chemical  analysis  is  more  rapidly  accomplished  than 
the  other  procedures,  and  was  formerly  regarded  as  a 
sufficient  basis  for  diagnosis  of  a  water  sample,  in  regard 
to  its  wholesomeness  or  otherwise  for  domestic  purposes. 
This  is  so  no  longer,  because  it  is  recognized  that  the  consti- 
tuents sought  for  and  actually  found  in  a  particular  water 
sample,  for  the  most  part  are  of  themselves  non-deleterious, 
and  by  their  excess  or  deficiency  simply  suggest  the  pre- 
sence or  absence  of  the  actual  materies  morbi.  Chemical 
evidence,  therefore,  must  be  used  in  conjunction  with  all 
the  other  evidence  before  a  definite  opinion  is  formed,  and 
even  then  the  judgment  may  be  wholly  based  on  negative 
findings,  which  here,  as  elsewhere,  may  at  any  time  not 
bear  the  interpretation  put  upon  them.  Nevertheless, 
the  following  statements,  when  cautiously  used,  are  helpful 
in  interpreting  results. 

High  chlorine  and  oxidized  nitrogen,  associated  with 
marked  free  and  albuminoid  ammonia,  suggest  present  or 
recent  animal  pollution. 

High  chlorine  and  oxidized  nitrogen  (not  from  strata), 
with  little  free  and  albuminoid  ammonia,  suggest  past  or 
remote  animal  pollution. 

Low  chlorine  and  oxidized  nitrogen,  and  very  low  free 
and  saline  ammonia  with  high  albuminoid  ammonia,  suggest 
pollution  of  vegetable  origin. 

Deep  wells  often  show  a  large  amount  of  chlorides  and 


WATER    ANALYSIS 


55 


free  ammonia,  without  these  necessarily  indicating  pollution. 
This  is  especially  notable  in  wells  sunk  into  strata  like  the 
London  greensands. 

The  organic  nitrogen  in  a  water  is  mostly  determined 
by  the  estimation  of  the  albuminoid  ammonia  which  only 
partially  measures  it.  If  the  organic  nitrogen  by  Frank- 
land's  process  be  I  part  per  100,000,  then  the  albuminoid 
ammonia  of  the  same  water  would  be  about  0*615  Part  Per 
100,000,  containing  0*506  of  organic  nitrogen ;  and  the 
organic  nitrogen  by  the  Kjeldahl  process  would  be  about 
double  that  in  the  albuminoid  ammonia,  or  1-012  part 
per  100,000. 


Specimen  An 

alyses  (from  Notter  and  Firth) 

Chlor- 
ine 

Free 
NH8 

All). 
NH3 

Oxygen 
absorbed 

Nitrates 

Nitrites 

1.   Upland  surface    ... 

10 

0  003 

0012 

0  290 

016 

nil 

2.   Shallow  well 

2*2 

0011 

0  009 

0  200 

0  002 

nil 

3. 

10 

nil 

0  003 

0  040 

0  800 

nil 

4. 

125 

0005 

0  006 

0150 

1500 

traces 

5.  Deep  well 

2'8 

0010 

0  004 

0  060 

0030 

nil 

6.       „        „ 

290 

0055 

0  002 

0110 

0110 

nil 

7.        M       „ 

190 

0018 

0  004 

0110 

0390 

traces 

8.       ,, 

220 

0011 

0  004 

0  060 

0  090 

nil 

9.   Spring  in  a  copse... 

1*6 

0020 

0001 

0015 

rioo 

nil 

10.         ,,      near  ditch 

40 

nil 

0  006 

0  200 

1700 

nil 

11.         ,,      in  meadow 

3'9 

0008 

0030 

0180 

0  200 

nil 

12.         ,,      protected... 

30 

0  009 

0  006 

0122 

0  600 

nil 

Notes. 
Local  Inspection  : 

In  numbers  1,  2,  5,  8,  and  12,  sources  of  pollution  were  absent 

or  well  guarded  against. 
Numbers  3  and  7  were  in  farmyards. 

In  numbers   4,  6,  9,    10,    and    11,    defects    in   construction    or   in 
protection  from  possible  pollution  were  found. 

Bacteriological  Examination  : 

In  numbers  4,  6,  7,   10,  and  11,  sewage  organisms  were  found. 

Opinion  : 

Numbers  1,  3,  5,  8,  and  12  were  returned  as  safe;    number  2  as 
doubtful;    and  numbers  4,   6,  7,  9,    10,   and   11,   as  unsafe. 


56 


PUBLIC    HEALTH    CHEMISTRY 


SEWAGE  AND  SEWAGE  EFFLUENTS. 

The  subjoined  table  will  suggest  ideas  on  this  subject. 


M  2 

C5 


c«s 


3<C 


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I    I    I   I   I    I 


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I    I 


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5  o   I    l 

3 


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p 


i    i    i 


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M     O     O 


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o  w 


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H 

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gooo 


WATER    ANALYSIS  57 

Examination  of  a  Sample. 

Equal  quantities  are  taken  every  hour  and  mixed,  and  a 
sample  is  then  taken.  This  is  examined  for  the  following 
constituents  and  characteristics.  The  bottle  containing  the 
sample  should  be  completely  filled,  and  examined  at  once 
or  kept  in  an  ice  chamber. 

Chlorine. — Dilute  with  as  much  distilled  water,  and 
examine  as  in  water  analysis. 

Ammonias. — In  a  crude  sewage  or  unfiltered  effluent, 
5  c.c.  should  be  diluted  to  500  c.c,  but  in  a  good  effluent  a 
dilution  of  ten  times  will  usually  be  sufficient.  Test  a 
little  with  Nessler,  and  judge  from  coloration  produced. 

Oxygen  Absorption. — Dilute  ten  to  one  hundred  times, 
and  be  careful  to  watch  for  decolorization  of  the  perman- 
ganate, as  several  lots  may  be  needed. 

Nitrates  and  Nitrites  are  estimated  as  in  water  ;  but  as 
seen  in  the  table  above,  nitrates  may  be  present  in  large 
amount,  especially  in  a  good  effluent,  and  the  coloration 
produced  will  be  much  stronger  than  that  in  the  standard. 

Suspended  Solids  can  be  estimated  by  filtering  a  known 
quantity  of  the  sample  through  a  weighed  filter  paper,  and 
drying  and  weighing  again.  The  filter  paper  is  then 
ignited  in  a  weighed  platinum  or  other  crucible,  which  is 
cooled  and  weighed.  The  increase  in  weight  of  the  crucible, 
less  the  weight  of  filter-paper  ash,  gives  the  mineral  -content 
in  the  suspended  solids.  The  loss  or  difference  between 
the  total  and  the  mineral  part  is  the  amount  of  organic 
matter  in  suspended  solids. 

Dissolved  Solids  are  estimated  in  the  filtrate. 

Incubator  Test  is  a  test  for  putrescibility  laid  down  by 
the  Mersey  and  Irwell  Joint  Committee  for  effluents  dis- 
charged into  the  Manchester  Ship  Canal.  The  effluent  is 
tested  in  the  fresh  state  for  oxygen  absorption  in  three 
minutes.  A  bottle  is  then  completely  filled  with  the 
sample,  stoppered,  and  incubated  at  8o°  F.  (270  C.)  for  a 
week.  The  contents  are  thereafter  tested  for  oxygen 
absorption  in  three  minutes.  If  the  amount  absorbed  is 
the  same  or  less,  the  effluent  is  considered  harmless  as 
regards  its  power  of  absorbing  oxygen  from  any  stream 
or  river  into  which   it   may  be  poured.     If  the  oxygen 


58  PUBLIC    HEALTH    CHEMISTRY 

absorption  has  increased,  putrefaction  is  inferred,  and  the 
effluent  is  considered  unsuitable.  Less  absorption  may  be 
noted,  due  to  oxidation  having  taken  place  at  the  expense 
of  the  nitrates  and  the  dissolved  oxygen. 

Dissolved  Oxygen. — Winkler's  process,  given  under 
water,  was  stated  to  be  unsuitable  in  the  presence  of 
much  organic  matter.  It  is,  however,  the  method  used 
by  the  Glasgow  Corporation  Chemist  for  the  effluents 
submitted  to  him  daily  for  analysis.  The  interference  of 
the  organic  matter  is  got  rid  of  by  the  addition  of  a  few 
drops  of  weak  permanganate  solution,  until  a  faint  pink 
colour  remains  permanent.  The  process  is  then  proceeded 
with  as  before.  Another  process  described  is  that  of  Letts 
and  Blake.  In  it  a  solution  of  ferrous  sulphate  is  added 
to  a  measured  quantity  of  the  sample  contained  in  a  bulb 
having  two  openings,  one  closed  with  a  stopper,  and  the 
other  leading  to  a  smaller  bulb  from  which  it  is  separ- 
ated by  a  stopcock.  Some  ammonia  is  added,  which 
precipitates  ferrous  hydrate.  This  absorbs  any  dissolved 
oxygen  in  the  sample,  becoming  ferric  hydrate.  Sulphuric 
acid  in  excess  is  then  added,  which  dissolves  the  two 
hydrates,  forming  the  corresponding  sulphates,  which  being 
more  stable  allow  the  end  titration  to  be  done  in  an  open 
vessel  without  risk  of  further  oxidation.  The  amount  of 
ferrous  sulphate  taken  is  titrated  with  standard  potassium 
permanganate  solution,  or  standard  potassium  bichromate 
solution,  each  of  which  is  of  such  a  strength  that  I  c.c.  = 
i  c.c.  oxygen.  This  preliminary  titration  measures  the 
amount  of  oxygen  required  to  oxidize  the  ferrous  sulphate, 
and  the.  end  titration  with  one  of  the  same  solutions 
measures  the  amount  of  ferrous  salt  still  unoxidized,  and 
the  difference  between  the  two  titrations  gives  the  number 
of  c.c.  of  oxygen  supplied  by  the  quantity  of  sample  taken, 
that  is,  the  amount  of  dissolved  oxygen.  Nitrites  have  a 
very  disturbing  effect  on  the  process,  and  to  obviate  this 
2  c.c.  of  a  mixture  of  3  volumes  permanganate  solution  and 
1  volume  50  per  cent  sulphuric  are  added,  and  the  sample 
is  allowed  to  stand  ten  minutes  before  proceeding.  The 
preliminary"  titration  of  the  ferrous  sulphate,  mixed  with 
the  same  amount  of  sample  and  sulphuric,  is  similarly 
treated. 


WATER    ANALYSIS  59 

Solutions  required  :  (a)  Ferrous  sulphate  solution  (5  per 
cent  in  1  per  cent  H2S04)  ;  (b)  Standard  potassium  per- 
manganate solution  (5-638  grm.  per  litre),  1  c.c.  =  1  c.c. 
oxygen  at  normal  temperature  and  pressure  ;  (c)  Ltd. 
pot.  bichromate  solution  (879  grm.  per  htre),  1  c.c.  =  1  c.c. 
oxygen  at  normal  temperature  and  pressure ;  (d)  Sulphuric 
acid,  50  per  cent  in  distilled  water. 

Process. — Measure  the  exact  capacity  of  the  large  bulb. 
Fill  it  by  siphonage  to  avoid  absorption  of  atmospheric 
oxygen.  Insert  stopper  and  drain  off  excess  of  fluid. 
Remove  stopper,  withdraw  7  c.c.  of  fluid,  add  5  c.c:  of 
ferrous  sulphate  solution  by  pipette  to  bottom  of  bulb, 
fill  up  mouth  with  strong  ammonia  solution,  and  insert 
stopper.  Mix  by  swinging.  A  precipitate  of  greenish 
ferrous  hydrate  forms,  which  absorbs  dissolved  oxygen, 
becoming  brownish  ferric  hydrate.  Allow  to  stand  fifteen 
minutes.  Then  add  50  c.c.  of  the  sulphuric  acid  solu- 
tion to  the  small  bulb,  and  opening  the  stopcock 
allow  it  to  mix  into  the  larger  bulb.  The  hydrates  are 
dissolved,  and  sulphates  formed.  Pour  out  into  a  beaker, 
and  titrate  with  permanganate  in  case  of  water,  and  with 
bichromate  for  sewage.  For  latter,  end  reaction  is  got 
by  removing  a  drop  of  liquid  from  the  beaker,  and  touch- 
ing a  drop  of  solution  of  potassium  ferricyanide,  when  if 
oxidation  is  incomplete,  a  blue  colour  (Turnbull's  blue)  is 
produced  within  two  minutes.  A  control  or  preliminary 
titration  is  made  by  taking  the  same  quantity  of  sample 
(less  7  c.c),  adding  first  50  c.c.  of  the  sulphuric  acid,  then 
5  c.c.  of  the  ferrous  sulphate  solution,  and  then  titrating. 
The  end  of  the  titration  with  permanganate  is  a  permanent 
faint  pink  tint. 

The  Sewage  Commission  recommend  that  the  suspended 
solids  should  be  removed  before  estimating  the  dissolved 
oxygen,  because  "  small  variations  in  the  amounts  of  solids 
in  suspension  in  effluents  may  seriously  affect  the  rate  at 
which  effluents  take  up  oxygen." 

Kjeldahl's  Method  for  Total  Nitrogen. — Owing  to  the 
large  amount  of  organic  matter  in  sewage,  the  estimation 
of  albuminoid  ammonia  is  alleged  to  be  unreliable,  and 
the  estimation  of  the  total  nitrogen  is  preferred. 


60      PUBLIC  HEALTH  CHEMISTRY 

Process. — Take  a  Kjeldahl  flask  (a  200  c.c.  flask  with 
a  round  bottom,  and  made  of  fire-resisting  glass),  pipette 
into  it  10  c.c.  of  sewage  or  25  c.c.  of  the  effluent.  Add 
•1  c.c.  of  strong  sulphuric  acid,  mix  well,  and  evaporate 
slowly  over  a  small  flame  guarded  with  wire  gauze.  When 
the  fluid  is  reduced  to  very  small  bulk,  add  5  grm. 
potassium  sulphate,  and  20  c.c.  strong  sulphuric  acid. 
Heat  over  a  small  Bunsen  flame,  very  slowly  at  first,  until 
all  frothing  ceases.  Continue  heating  for  about  two 
hours,  or  until  the  liquid  is  colourless  and  clear.  Then 
cool  the  flask  and  contents,  and  carefully  transfer  the 
liquid  to  a  700  c.c.  boiling-flask,  washing  the  small  flask  out 
repeatedly  with  ammonia-free  distilled  water,  and  adding 
the  washings  to  the  large  flask,  making  up  the  bulk  to 
about  200  c.c.  Now  add  sufficient  strong  KOH  solution 
to  neutralize  the  excess  of  acid,  and  some  extra  to  make 
alkaline.  Add  a  few  pieces  of  granulated  zinc  to  prevent 
bumping,  connect  with  a  Kjeldahl's  safety  bulb  and  con- 
denser, and  distil  over  the  ammonia  into  N/i  sulphuric 
acid,  the  end  of  the  tube  from  the  special  condenser  dipping 
under  the  surface  of  20  c.c.  of  the  N  /i  acid  in  an  Erlenmeyer 
flask.  About  100  c.c.  are  distilled  over,  slowly.  There- 
after the  acid  is  titrated  with  N/i  NaOH,  using  methyl- 
orange  as  indicator.  The  number  of  c.c.  of  soda  required 
deducted  from  the  number  of  c.c.  of  normal  acid  used  (20) 
gives  the  number  of  c.c.  of  normal  acid  neutralized  by 
the  ammonia  distilled  over.  Each  c.c.  of  normal  acid  is 
chemically  equivalent  to  0-014  grm.  of  N.  The  organic 
N  is  got  by  deducting  from  the  result  obtained  the  amount 
previously  found  to  be  present  as  free  and  saline  ammonia. 
(When  this  process  is  applied  to  food  stuffs,  this  correction 
is  not  necessary.) 

The  rationale  of  the  process  is  that  the  organic  matter 
is  broken  up  by  the  acid  and  sulphate,  and  the  nitrogen 
converted  into  ammonia,  which  is  fixed  by  the  excess  of 
acid  as  ammonium  sulphate.  On  adding  excess  of  KOH, 
the  ammonia  is  liberated,  and  is  then  distilled  over.  It  is 
received  into  N/i  sulphuric  acid,  forming  again  ammonium 
sulphate.  The  amount  of  acid  left  unneutralized  is  then 
estimated,  and  by  difference  the  amount  combined  with 
the  ammonia. 


WATER    ANALYSIS 


61 


Glasgow  Sewage. — Average  of  the  daily  analyses  of 
sewage  treated  at  Dalmarnock  and  Dalmuir  sewage  works 
for  the  year  ending  May,  1910  ;  and  of  the  unfiltered 
effluents  discharged  after  treatment  by  precipitation 
processes.     In  parts  per  100,000. 


Dalmarnock 

Dalmuir 

Crude 
Sewage 

Effluent 

Crude 
Sewage 

Effluent 

Suspended  solids : 

Total 

47-2 



250 



Organic 

23-4 



I4I 



Mineral 

23'8 



109 



Chlorine 

21*4 

200 

70 

7'2 

Free  and  Saline  Ammonia . . 

1-89 

1-18 

1  94 

I  60 

Albuminoid  Ammonia 

•522 

•3°5 

•407 

•263 

Oxygen  absorbed  in  4  hrs. 

at  8o°  F 

6-818 

2-617 

4610 

2-837 

Per  cent,  purification — 

On  Free  and  Saline  NH3  .  . 

— • 

37-5  % 

— 

175% 

On  Albuminoid  NH3 

— 

4i-5  % 

— 

35  3% 

On  Oxygen  absorbed 

— 

61  -6  % 

38-4% 

CHAPTER    III. 
EXAMINATION     OF     AIR. 

The  chief  points  to  determine  are  odour,  temperature, 
pressure,  humidity,  carbonic  acid,  ozone,  oxidizable  and 
organic  matter,  noxious  emanations,  micro-organisms, 
suspended  matter,  carbon  monoxide,  oxygen. 

Examination   of  a  Sample. 

Collection. — Large  wide-mouthed  jars  with  rubber  caps, 
and  holding  about  4000  c.c.  (4  litres),  are  most  convenient. 
These  are  thoroughly  cleansed  with  distilled  water  before 
use,  run  dry  by  inverting,  and  capped.  Their  actual 
capacity  should  be  ascertained,  and  marked  on  them.  To 
collect  a  sample  of  air,  one  of  two  methods  may  be 
employed,  namely  : 

1.  Place  jar  where  sample  is  to  be  taken,  and  blow  in 
the  surrounding  air  by  a  pair  of  bellows  having  a  long 
nozzle  reaching  down  to  the  bottom  of  the  jar.  The 
contained  air  is  thus  displaced  from  the  jar. 

2.  Fill  the  jar  with  distilled  water,  and  empty  it  at  the 
place  named  for  sampling  by  inverting  it,  and  allowing  it 
to  drain  dry.  It  is  then  capped  and  labelled,  and  the  label 
inscribed  with  the  observed  temperature  and  pressure  and, 
if  not  already  noted,  the  capacity  of  the  jar. 

Great  care  must  be  exercised  not  to  contaminate  any 
sample  with  the  air  breathed  out  by  the  observer. 

Odour. — The  sense  of  smell  exceeds  in  acuteness  any 
other  means  used  at  present  to  demonstrate  the  presence 
of  minute  particulate  matter.  It  also  has  a  special  value 
in  detecting  the  peculiar  fcetid  odour  so  noticeable  on  first 
entering  an  occupied  room  from  the  open  air.  De 
Chaumont  was  the  first  to  emphasize  this,  and  he  further 
pointed  out  the  importance  of  observing  it  immediately 
on  entering,  as  the  sense  of  smell  is  soon  blunted.  He 
further  pointed  out  the  marked  influence  of  atmospheric 
humidity  in  rendering  the  smell  of  organic  matter  more 
perceptible,   an  increase  of  1  in  the  humidity  being  as 


EXAMINATION    OF    AIR  63 

powerful  in  this  respect  as  a  rise  of  2-32°  C.  or  4-18°  F.  (see 
Gordon  Report  on  Ventilation  of  Houses  of  Parliament," 

I9°5). 

Temperature. — By  the  thermometer. 

Pressure. — By  the  barometer  fitted  with  a  vernier 
(e.g.  Fortin's  standard  barometer). 

Humidity. — By  the  hygrometer  (direct  or  indirect). 
Daniell's,  Regnault's,  Dines',  Mason's  (wet  and  dry  bulb). 
Absolute  humidity,  relative  humidity,  dewpoint,  Glaisher's 
factors,  Apjohn's  formula. 

Carbonic  Acid  Gas. — The  determination  of  CO  2  affords 
an  important  index  as  to  the  extent  to  which  other  impuri- 
ties exist.  It  is  usually  estimated  by  Pettenkofer's 
method.  Solutions  required  :  (a)  Baryta  water  0-5  per 
cent  ;  (b)  Standard  oxalic  acid  solution  1  c.c.  =  0-5  c.c. 
CO  2  at  normal  temperature  and  pressure  (2-822  grm.  per 
litre). 

Process. — This  consists  in  exposing  a  measured  quantity 
of  baryta  water  to  a  known  volume  of  air  enclosed  in  a  jar. 
The  baryta  water  absorbs  the  carbonic  acid  gas,  becoming 
barium  carbonate,  which  is  precipitated.  The  amount  of 
baryta  water  unused  or  unchanged  is  estimated  by  titra- 
tion of  a  measured  portion  of  the  baryta  water  removed 
from  the  jar,  with  standard  solution  of  oxalic  acid  in  the 
presence  of  phenolphthalein.  Oxalic  acid  is  used  in  prefer- 
ence to  sulphuric  acid,  because  the  latter  would  attack 
particles  of  barium  carbonate  floating  in  the  liquid,  and 
thus  give  rise  to  some  degree  of  error. 

BaH„02  +  C02  =  BaC03  +  H20 

BaH  20  2  +  H  2C  20  4-2H  20  =  BaC  20  4  +  4'H  20. 

From  these  equations  we  see  that  one  molecule  of  baryta 
water  is  neutralized  by  one  molecule  of  C02  and  one 
molecule  of  oxalic  acid.  Hence  the  two  latter  are  chemi- 
cally equivalent,  and  1  molecule  of  oxalic  acid  measures 
indirectly  1  molecule  C02;  that  is,  126  grm.  (cryst.) 
oxalic  measure  indirectly  44  grm.  C02,  or  126  grm. 
(cryst.)  oxalic  measure  22-32  litres  C02,  or  2-822  grm. 
oxalic  measure  0-5  litre  CO  2.  Hence,  if  we  dissolve  2-822 
grm.  of  crystallized  oxalic  acid  in  1  litre  of  distilled 
water,  1  c.c.  =  0-5  c.c.  CO  2. 


64  PUBLIC    HEALTH    CHEMISTRY 

Methods. — A  large  jar  is  filled  with  the  air  sample  as 
directed  above.  Fifty  c.c.  of  the  baryta  water  are  added, 
the  jar  is  capped,  and  shaken  now  and  then  over  a  period 
of  half  an  hour.  Thereafter  25  c.c.  are  removed  from  the 
jar  and  titrated  with  the  standard  oxalic,  using  phenol- 
phthalein  as  indicator  until  the  red  colour  is  just  discharged. 
Twenty-five  c.c.  of  fresh  baryta  are  similarly  titrated,  and 
the  number  of  c.c.  required  noted.  The  difference  between 
this  latter  and  the  number  of  c.c.  required  for  the  25  c.c. 
removed  from  the  jar,  measures  in  oxalic  the  amount  of 
baryta  which  has  had  its  alkalinity  neutralized  by  absorp- 
tion of  CO  2  from  the  air  in  the  jar.  This  amount  doubled 
measures  the  quantity  so  neutralized  in  the  50  c.c.  taken, 
and  as  the  oxalic  per  c.c.  =  0-5  c.c.  CO  2,  on  multiplying 
the  result  by  0-5  we  get  the  number  of  c.c.  of  CO  2  absorbed 
from  the  volume  of  air  in  the  jar.  The  C02  is  at  normal 
temperature  and  pressure,  and  the  air  in  the  jar  is  at  the 
temperature  and  pressure  noted  on  collection  of  the  sample, 
and  so  these  two  volumes  are  not  quite  comparable.  The 
volume  of  sample  in  the  jar  must  first  be  corrected  to  the 
volume  it  would  occupy  at  standard  temperature  and 
pressure.  This  may  be  done  after  several  fashions,  but 
the  one  here  recommended  is  to  use  the  formula 

V°  X  P°       V  X  P' 


'po  qp/ 

where  V°  P°  and  T°  denote  respectively  volume  at 
standard  pressure  (760  mm.  or  29-92  inches  of  mercury) 
and  at  standard  absolute  temperature  (o°  C  +  273  or 
320  F.  -f  459),  and  V,  P',  and  T',  the  volume  of  the  jar  as 
measured  (Jess  50  c.c.  displaced  by  the  baryta  water 
added),  and  the  pressure  and  temperature  at  the  time  and 
place  of  taking  the  sample,  the  temperature  being  changed 
to  the  absolute  scale. 

Thereafter  the  proportion  of  C02  present  is  calculated 
and  the  result  expressed,  which  may  be  stated  as  a  per- 
centage, or  parts  per  1000  or  per  10,000.  The  air  of  the 
open  country  averages  almost  exactly  3  parts  per  10,000. 

Haldane's  Method  requires  a  special  apparatus,  but  once 
facility  of  manipulation  of  the  apparatus  has  been  acquired, 
an  accurate  result  can  be  obtained  in  ten  minutes  and 


EXAMINATION    OF    AIR  65 

without  any  calculation.  The  method  consists  in  subjecting 
25  c.c.  of  air  to  exposure  to  caustic  potash,  which  absorbs 
the  CO  2,  and  the  diminution  in  volume  is  measured  under 
the  same  conditions  of  temperature  and  pressure,  and  the 
divisions  on  the  graduated  portion  of  the  burette  are  each 
too  00 *h  Pat"t  °f  the  whole  capacity  of  the  burette,  so  that 
the  result  is  read  off  in  parts  per  10,000. — 2KOH  -f-  CO  2  = 
K2C03  +  H20. 

Hesse's  Modification  of  Pettcnkofer's  Method  is  to  collect 
the  sample  in  a  flask,  from  250  c.c.  to  1000  c.c.  capacity, 
closed  with  a  rubber  stopper  with  two  holes  plugged  with 
glass  rods.  One  of  the  plugs  is  removed,  and  a  pipette 
containing  10  c.c.  of  baryta  is  inserted  in  its  place.  The 
other  plug  is  loosened,  and  the  baryta  allowed  to  run  into 
the  flask.  The  pipette  is  then  removed  and  the  plugs  are 
reinserted,  and  the  flask  shaken  from  time  to  time.  After 
half  an  hour  a  plug  is  withdrawn,  a  drop  of  phenol- 
phthalein  added,  and  the  nozzle  of  a  burette  containing 
standard  oxalic  one-tenth  the  strength  of  that  used  in 
Pettenkofer's  process  is  placed  in  the  vacant  hole,  and  the 
baryta  in  the  flask  titrated.  The  other  titration  to  ascer- 
tain the  oxalic  equivalent  of  10  c.c.  of  fresh  baryta  is 
done  as  before.  The  advantage  of  the  method  is  that 
there  is  less  exposure  of  the  baryta  to  the  air  not  in  the 
flask.     Equations — 

Ba(OH)  2  +  CO  2  =  BaCO  3  +  H  20 
Ba(OH)  2  +  H  2C  20  4  =  BaC  20  4  +  2H  20. 

Pettenkofer's  name  is  attached  to  another  method  in 
which  air  is  aspirated  through  a  known  bulk  of  baryta 
water  spread  out  lengthwise  in  a  tube  so  that  the  air 
bubbles  through  the  baryta  solution. 

Lunge  and  Zeckendorfs  Method  is  to  take  a  known 
quantity  of  N/500  Na2C03  in  a  glass  bottle  fitted  with  a 
two-holed  stopper  and  two  glass  tubes  as  in  a  wash-bottle. 
An  indiarubber  bulb  of  a  standard  capacity  with  an  inlet 
and  an  outlet  tube,  both  fitted  with  valves,  has  its  outlet 
tube  attached  to  the  glass  tube  leading  to  the  bottom  of 
the  bottle  and  under  the  surface  of  the  solution  of  sodium 
carbonate.  The  bulb  is  compressed  slowly,  and  the  air 
expressed  bubbles  through  the  fluid  in  the  bottle,   and 


66  PUBLIC    HEALTH    CHEMISTRY 

the  latter  is  then  shaken.  The  manoeuvre  is  repeated 
until  the  fluid  (which  had  been  coloured  pink  by  the  addi- 
tion of  phenolphthalein)  becomes  colourless  from  the 
Na2C03  absorbing  C02  and  forming  NaHC03.  The 
number  of  compressions  of  the  bulb  required  are  counted, 
and  by  reference  to  a  table  the  proportion  of  carbonic 
acid  gas  present  in  the  air  tested  is  obtained.  The  method 
is  very  tedious  if  the  air  is  pure,  as  forty-eight  compres- 
sions are  required  to  detect  3  parts  per  10,000. 

Scurfield's  Apparatus. — Air  is  aspirated  through  weak 
baryta  solution,  3  parts  per  10,000,  coloured  red  by 
addition  of  phenolphthalein. 

Ozone. — 

1.  Houzeaus  paper.  A  piece  of  neutral  or  faintly 
reddened  litmus  paper  soaked  for  a  quarter  of  its  length 
in  neutral  KI  solution  and  dried  (keep  in  dark).  When 
exposed  to  ozone  the  KI  is  decomposed  and  K20  formed, 
which  turns  the  litmus  blue  at  soaked  part  only.  CI  and 
Br  would  not  do  this.  N  20  3  would  turn  rest  red.  NH  3 — 
use  control. 

2.  Schonbeins  paper.  KI  and  starch  paper  turned  blue. 
CI,  Br,  N  20  3,  H  20  2  all  give  same  result. 

3.  KI  and  phenolphthalein  papers  turned  red.  H202 
gives  same  result.     CI,  Br,  and  N  20  3  give  a  brown. 

4.  Arnold-Mentzel  paper.  Chromic  acid  paper  is  negative. 
Hydroxyl  gives  a  blue. 

5.  Engler  and  Wild's  manganous  chloride  paper.  Ozone 
turns  it  brown.  Hydroxyl  has  no  action,  but  ammonia 
also  gives  a  brown. 

Oxidizable  and  Organic  Matter.  —  This  is  best 
determined  by  washing  the  air  (by  shaking  with  distilled 
water  or  slowly  aspirating  through  the  same)  and  examin- 
ing the  washings,  as  in  water  analysis,  for  total  nitrogen- 
free,  saline  and  albuminoid  ammonias,  nitrous  and  nitric 
acids,  and  oxygen  absorption. 

Carnelly's  process  is  sometimes  used,  but  is  not  very 
reliable.  A  millinormal  solution  of  permanganate  is  acidu- 
lated with  sulphuric  acid,  and  50  c.c.  are  added  to  an  air 
jar  containing  the  sample,  and  exposed  with  occasional 
shakings  to  its  influence  for  about  half  an  hour.     There- 


EXAMINATION    OF    AIR  67 

after  25  c.c.  are  removed  and  compared  with  the  same 
quantity  of  fresh  solution,  and  the  number  of  c.c.  of 
same  required  to  be  added  to  bring  its  colour  up  to  that  of 
control  is  noted.  From  this  number  the  amount  of  oxygen 
absorbed  can  be  calculated  and  the  result  expressed  in 
parts  per  million. 

Noxious  Emanations. — Under  this  heading  is  con- 
sidered the  search  for  foreign  gases  and  vapours  in  the 
air,  such  as  fumes  of  hydrochloric,  nitric  and  nitrous, 
carbonic,  and  sulphurous  acids,  sulphuretted  hydrogen, 
chlorine,  ammonia,  carbon  monoxide,  ammonium  sulphide, 
carbon  bisulphide,  carburetted  hydrogen,  roburite,  nitro- 
benzol. 

Scheme  for  detection. — Take  sample  in  jar. 

1.  Remove  cap  or  stopper,  smell,  and  replace  stopper. 
Chlorine,  HC1,  S02,  ammonia,  ammonium  sulphide, 
sulphuretted  hydrogen  and  carbon  bisulphide,  all  have 
characteristic  odours.  Carbonic,  nitrous,  and  nitric  acids 
have  not. 

2.  Take  a  piece  of  red  and  a  piece  of  blue  litmus  paper  ; 
moisten  in  some  neutral  distilled  water,  attach  to  a  piece 
of  stick,  and  hang  down  into  jar  free  of  sides.  After  waiting 
a  minute,  note  change  of  colour. 

3.  If  reaction  acid  or  alkaline,  pour  rapidly  into  jar 
10  c.c.  of  ammonia-free  distilled  water,  replace  stopper, 
and  shake  vigorously.  Remove  half  of  this  water,  and 
test  for  dissolved  gas. 

A.  If  the  reaction  was  acid,  then  it  is  likely  to  be 
carbonic,  hydrochloric,  sulphurous,  nitric,  or 
nitrous  acid.  Add  to  water  removed  from  jar  a 
few  drops  of  silver  nitrate  solution. 

White  precipitate  denotes  either  : — 

a.  Carbonic  acid  :  precipitate  very  slight, 
acidity  also  very  faint,  baryta  water  added 
to  jar  becomes  turbid  after  shaking,  and 
turbidity  is  increased  by  adding  ammonia. 

b.  Hydrochloric  acid :  precipitate  marked, 
acidity  ditto,  precipitate  insoluble  in  nitric 
acid,  soluble  in  ammonia,  and  also  in  KCN. 


6  8  PUBLIC    HEALTH    CHEMISTRY 

c.  Sulphurous  acid :  precipitate  marked, 
soluble  in  nitric,  soluble  also  on  heating, 
but  the  solution  darkens  from  formation 
of  sulphide  of  silver.  The  water  will  also 
decolorize  iodide  of  starch  solution,  and 
warmed  with  Zn  and  HC1  gives  off  H2S, 
which  darkens  lead  acetate  paper.  Odour 
charact  eristic. 

No  precipitate,  infer  nitric  or  nitrous  acid. 
Test  for  these  as  described  under  water 
analysis. 

B.  If  the  reaction  was  alkaline,  gas  is  either  ammonia 
or  Am2S. 
To  water  from  jar  add  a  little  Nessler's  solu- 
tion.    Yellow  to  amber  colour — ammonia. 

Characteristic  odour. 
Black  colour,  Am  2S  ;   nitroprusside  of  Na 
gives  a  violet  ;    smell  of  H  2S. 

4.  If  litmus  is  unaffected,  may  be  either  : — 

H  2S,  PbAc  papers  blackened,  odour  characteristic. 
CS2,     colourless     volatile     liquid     giving     off     an 

inflammable    vapour   with    a   garlicky    odour. 

The  liquid  burns  with  a  blue  flame  giving  off 

sulphur  dioxide  fumes,  and  leaving  a  deposit 

of  sulphur. 

5.  If  the  blue  litmus  is  first  slowly  reddened  and  then 
bleached,  the  gas  is — 

Chlorine  :  filter  paper  moistened  in  KI  solution  is 
first  darkened  and  then  bleached.  Odour 
characteristic.  Red  colour,  with  mixture  of 
proto-salt  of  iron  and  KCNS. 

Estimation  of  some  Gases  detected  as  above. — Hydrochloric, 
nitrous,  and  nitric  acids  are  absorbed  in  freshly  distilled 
water,  and  tested  for  as  in  water  analysis.  Chlorine  by 
pure  KI  solution,  from  which  it  liberates  iodine,  which  is 
titrated  with  thiosulphate  of  sodium.  Bromine  similarly. 
Sulphurous  acid  by  absorption  in  a  decinormal  solution  of 
iodine.  Sulphuretted  hydrogen  similarly.  Carbon  bi- 
sulphide by  absorption  in  a  strong  solution  of  potash  in 
alcohol,  and  after  titration  with  standard  iodine  solution. 


EXAMINATION    OF    AIR  69 

In  all  these  cases  the  air  to  be  tested  is  slowly  aspirated 
through  the  liquid  named.  Ammonia  can  be  absorbed  in 
pure  water  and  Nesslerized. 

Micro-organisms. — See  Bacteriology. 

Suspended  Matter. — Aspirate  large  quantities  of  air 
through  small  amounts  of  water  in  a  series  of  wash-bottles  ; 
evaporate  down  to  aliquot  part,  mount  a  drop  and  count 
number  of  particles,  or  to  dryness  and  weigh  residue,  which 
may  then  be  ignited  and  cooled  and  re-weighed  for  non- 
volatile part.  Pouchet's  aeroscope.  Hesse's  apparatus. 
Sugar  filter.  Aitken's  method.  Is  composed  of  animal, 
vegetable,  and  mineral  matter.  Varies  in  towns  from 
5  to  25  mgr.  per  cm.,  or  otherwise  expressed  from  10,000 
to  2,000,000  particles  per  c.c.  Shaw  calculates  that 
400  tons  of  soot  are  thrown  into  the  air  of  London  per 
day.  In  London  40  cwt.  of  soot  are  deposited  on  each 
acre  of  ground  per  annum,  and  in  Glasgow  22  cwt.  in 
summer  and  25  in  winter,  making  47  in  all. 

Carbon  Monoxide. — From  stoves,  in  water  gas,  6  per 
cent  in  coal  gas,  in  mines.  Affinity  for  haemoglobin  300 
times  that  of  oxygen.  Kills  when  blood  is  saturated  up 
to  60  to  80  per  cent.  Haldane  advises  the  use  of  birds 
and  mice  as  indicators  in  mines. 

Haldane  s  Method. — Take  5  c.c.  dilute  blood  solution  in 
a  clean  dry  bottle,  aspirate  in  some  suspected  air,  cork 
and  shake  for  ten  minutes,  protecting  from  light.  Pour 
out  into  a  test  tube  and  compare  with  some  of  original 
blood.  If  CO  present,  the  treated  blood  will  be  pink. 
The  test  is  made  quantitative  by  adding  carmine  solution 
to  the  normal  blood  until  tints  equal,  and  repeating  with 
normal  blood  saturated  with  coal  gas. 

Spectroscope :  spectrum  similar  to  OxyH,  but  not  reduced 
by  Am2S.  May  also  be  absorbed  by  copper  subchloride 
in  a  Hempel's  gas  burette. 

Oxygen. — This  may  be  estimated  by  combustion  with 
hydrogen,  or  by  absorption  in  an  alkaline  solution  of 
pyrogallic  acid,  or  by  absorption  by  nitric  oxide.  The 
two  latter  are  done  in  a  Hempel's  gas  burette,  and  in  the 
pyrogallic  method  the  carbonic  acid  is  also  absorbed  and 
has  to  be  separately  estimated  and  deducted.     In  these 


70 


PUBLIC  HEALTH  CHEMISTRY 


methods  care  must  be  taken  that  the  temperature  and 
pressure  do  not  vary  while  the  experiment  is  in  progress. 
Gases  in  Mines. — The  atmosphere  in  mines  is  liable  to 
become  dangerously  contaminated  by  noxious  gases 
issuing  from  the  coal  face,  such  as  sudden  discharges  of 
marsh  gas  from  accumulations  under  pressure  in  the  coal 
measures  (so-called  "  blowers  "),  by  accumulation  of  dust 
which  may  fire,  and  by  the  gases  resulting  from  fires  and 
explosions.  The  following  table  gives  the  principal  gases 
found  in  mines,  and  their  popular  and  other  names. 


Miner's  Name 

Composition 

Occurrence 

Remarks 

Black-damp 
or  Stythe 

A    mixture    of 
gases,    contain- 
ing 85  to  88% 
of  nitrogen 

In  coal  mines 

Does   not  sup- 
port   life,     nor 
combustion 

After-damp 

or 
Choke-damp 

co„ 

carbon   dioxide 
carbonic  acid 

In  coal  mines 
In  lead  mines 

do. 

White -damp 

CO 

carbon  monoxide 
carbonic    oxide 

In  coal  mines 

Very  poisonous 
and    explosive 

Fire-damp 

CH4 
marsh  gas,  me- 
thane,     carbu- 
retted  hydrogen 

In  coal  mines 

Highly  explosive 

— 

H2S 

sulphuretted 

hydrogen 

In  sulphur 
mines 

Very  poisonous 
Inflammable 

— 

Coal  dust 

In  coal  mines 

Highly 
inflammable 

CHAPTER    IV. 
SOILS. 

Soil  is  the  term  used  to  denote  that  portion  of  the 
earth's  crust  which  by  its  condition  or  properties  can 
affect  health.  It  is  conveniently  spoken  of  as  composed  of 
two  layers  :  (i)  an  upper  or  surface  soil,  and  (2)  a  deeper 
or  subsoil  layer.  The  upper  layer  contains  the  products 
of  the  decay  of  animal  and  vegetable  matter,  constituting 
mould  or  "  humus."  The  subsoil  layer  is  intermediate 
between  the  upper  layer  and  the  underlying  formations  or 
strata.  Both  layers  are  originally  derived  from  these 
deeper  layers  by  "  weathering,"  a  geological  term  which 
includes  all  those  forces  that  make  for  denudation  of 
surface. 

Examination  of  a  Sample. 

Ground  Air. — The  amount  of  air  in  soil  varies  with  its 
porosity,  and  its  state  in  regard  to  moisture.  Thus  a  dry 
porous  soil  may  contain,  if  loamy,  70  per  cent  ;  if  loose 
sand,  40  to  50  per  cent.  Such  air  is  collected  by  aspiration 
through  a  tube  leading  to  a  perforated  bulb,  sunk  into  the 
soil,  in  which  an  opening  has  been  made.  The  analysis  of 
ground  air  is  made  for :  CO  2  (increases  with  depth,  most 
in  summer  and  autumn,  least  in  winter  and  spring,  more  in 
impure  porous  soils  ;  varies  from  4  to  8  per  cent  in  January, 
to  8  to  24  per  cent  in  August)  ;  Moisture  :  85  per  cent ; 
Oxygen  :  21  to  18  per  cent.  The  amount,  in  a  quantity  of 
soil,  may  be  estimated  by  filling  a  burette  to  the  zero  mark, 
with  sample  of  soil,  and  connecting  the  nozzle  with  that 
of  another  burette  containing  50  c.c.  of  water,  by  a  piece 
of  rubber  tubing.  On  opening  the  stopcocks  and  raising 
the  burette  containing  the  water,  the  water  flows  into  the 
other  burette,  wetting  the  soil.  The  process  is  stopped 
when  the  water  reaches  the  zero  mark,  by  closing  the 
stopcocks.  The  loss  of  water  from  the  one  burette  is  a 
measure  of  the  amount  absorbed  by  the  soil  displacing  the 
contained  air. 


72  PUBLIC    HEALTH    CHEMISTRY 

Ground  Water — Is  divided  into  (i)  Moisture,  which  is 
the  water  present  along  with  ground  air,  simply  moistening 
the  particles  ;  and  (2)  Subsoil  water,  which  is  the  condition 
when  the  particles  and  their  interstices  are  full  of  water. 

The  amount  of  moisture  in  a  soil  is  estimated  by  drying 
a  weighed  portion  of  it ;  the  loss  of  weight  being  calculated 
as  moisture. 

The  level  of  the  subsoil  water  is  studied  by  digging  a  pit 
or  well,  and  observing  from  time  to  time  the  varying  levels. 

Soil  Temperature— Is  taken  at  the  depth  of  4  feet  by  a 
specially  sluggish  thermometer,  enclosed  in  a  protecting 
case.  The  soil  temperature  attains  its  maximum  in  July 
and  August. 

Soil  is  also  examined  chemically  for  total  nitrogen, 
phosphates,  sulphates,  nitrates,  and  peaty  acids.  An 
aqueous  extract  of  a  weighed  quantity  of  it  may  be 
examined  for  chlorides,  ammonias,  etc.  It  is  also  separated 
mechanically  into  particles  of  different  sizes,  and  into  clay, 
sand,  etc. 


CHAPTER     V. 

FOODS. 

EXAMINATION     OF     MILK. 

Average  composition  of  cow's  milk  :— Water,  8y  to  88  %  ; 
proteid,  3  to  3-5  %  ;  fat,  3-5  to  4-5  %  ;  sugar,  4  to  5  % ; 
mineral  matters,  07  %. 

Physical  Characters.  —  Placed  in  a  narrow  glass  it 
should  be  quite  opaque,  of  full  white  colour,  without 
deposit,  without  peculiar  smell  or  taste,  and  when  boiled 
it  should  not  change  in  appearance.  The  temperature 
should  be  taken. 

Reaction. — Should  be  slightly  acid,  or  neutral,  or  very 
feebly  alkaline.  Fresh  milk  is  sometimes  both  acid  and 
alkaline  to  indicators,  that  is,  amphoteric,  turning  red 
litmus  blue  and  turmeric  to  brown.  Strongly  alkaline  : 
cow  ill,  or  much  colostrum,  or  addition  of  sodium  carb. 
Strong  acidity  :  lactic  or  butyric  acids,  and  indicative  of 
retrograde  change. 

Cream. — Stand  100  c.c.  in  a  measure  for  twenty-four 
hours  in  a  still  atmosphere.  Read  off  proportion  of  cream. 
Should  be  T£y  to  -J^ ;  generally  about  T^.  Alderney  cows 
give  3^  to  ^V     Time  of  year  and  breed  to  be  considered. 

Specific  Gravity. — At  150  C.  or  6o°  F.  Varies  from 
1027  to  1034,  being  less  as  fat  is  greater.  The  specific 
gravity  is  raised  by  skimming  and  can  be  reduced  by 
adding  water,  so  that  this  factor  alone  is  not  a  reliable  index 
to  the  character  of  a  sample.  The  specific  gravity  falls 
i°  for  each  rise  of  io°  F.  above  6o°,  and  at  6o°  F.  there 
is  a  loss  of  30  of  specific  gravity  for  every  10  per  cent  of 
water  added. 

Three  methods — Specific  gravity  bottle,  lactometer,  and 
Westphal  balance. 

Total  Solids. —  Ought  not  to  be  below  11-5  per  cent  ; 
but  are  more  usually  12  to  13  per  cent.  Take  2  c.c.  of  the 
milk  in  a  flat  shallow  dish  of  known  weight.  Evaporate 
to  dryness  over  the  water-bath,  and  then  in  the  water-oven 


74  PUBLIC    HEALTH    CHEMISTRY 

for  half  an  hour,  and  weigh.  The  increase  in  weight  is  the 
amount  of  total  solids  in  2  c.c.  If  the  specific  gravity  of 
the  milk  is  known,  the  weight  of  2  c.c.  is  readily  calculated, 
and  the  percentage  of  total  solids  is  then  worked  out. 
Some  analysts  weigh  5  grm.  of  milk  into  the  dish,  and 
thus  avoid  using  the  specific  gravity. 

Ash. — The  dried  solids  are  incinerated  at  as  low  a  heat 
as  possible,  and,  on  cooling,  the  dish  is  reweighed  and  the 
percentage  calculated.  It  averages  in  normal  milks  about 
073,  and  should  not  fall  below  07.  Watering  makes  it 
less.  Test  ash  for  effervescence ;  if  marked,  suggests 
addition  of  a  carbonate. 

Fat. — This  is  a  very  important  determination.  Several 
methods. 

Werner-Schmidt  Method. — Ten  c.c.  of  the  milk  are 
pipetted  into  a  Stokes'  tube,  and  strong  HC1  is  added  to  the 
20  c.c.  mark.  The  mixture  is  now  heated  in  the  water- 
bath,  or  carefully  over  a  flame,  until  it  turns  a  brown 
colour.  Now  cool  the  tube  and  its  contents  in  water,  and 
then  add  ether  to  the  50  c.c.  mark.  Cork  the  tube  firmly, 
and  mix  the  contents  well  by  inverting  slowly  ten  to 
twelve  times.  Set  the  tube  down  in  an  upright  position  to 
allow  the  ether  to  separate  and  become  clear.  Pipette  off 
10  c.c.  or  15  c.c.  of  the  ether  into  a  weighed  platinum 
capsule,  or  other  dish,  evaporate  the  ether  on  water-bath 
at  6o°  C,  dry  at  ioo°  C,  cool  and  weigh.  The  increase  of 
weight  is  weight  of  fat  dissolved  in  the  amount  of  ether 
taken  by  pipette.  Now  observe  number  of  c.c.  of  ether 
still  in  the  tube,  plus  f  of  the  fluffy  layer,  separating  the 
clear  ether  from  the  dark  liquor.  Calculate  by  proportion 
amount  of  fat  dissolved  in  this  quantity  of  ether,  and  add 
the  result  to  former  ;  the  sum  gives  the  amount  of  fat 
derived  from  10  c.c.  of  milk  sample.  Express  result  as 
weight  of  fat  per  100  grm.  of  milk.  To  do  this  specific 
gravity  of  sample  must  be  observed. 

Example. — Twenty  c.c.  of  ether  removed  from  tube 
gave  a  residue  of  0-286  grm.  Ether  still  in  tube  (plus  f 
fluffy  layer)  2-8  c.c.  Therefore  as  20  :  22-8  :  :  0-286  :  x  — 
0-326  grm.  of  fat  in  10  c.c.  of  milk  sample.  But  specific 
gravity  of  sample  was  1030-5,  hence  10  c.c.  weigh  10-305 


FOODS  75 

grm.  Then  by  proportion — 10-305  :  100  :  :  0-326  :  x  = 
3-16  grm.  of  fat  in  100  grm.  of  milk,  or  3-16  per  cent. 

Adams'  Process. — Using  a  Soxhlet  apparatus  is  the 
official  method  of  the  Society  of  Public  Analysts. 

Take  a  strip  of  Adams'  fat-free  paper,  and  from  a  pipette 
spot  over  it  5  c.c.  of  milk  sample.  Dry  the  paper  high  over 
a  Bunsen  flame,  and  finally  in  the  water-oven.  Roll  paper 
up  into  a  coil,  and  put  it  into  a  Soxhlet  apparatus  attached 
below  to  a  clean  dry  flask  (wide-mouthed)  of  known  weight 
and  above  to  an  invert  condenser.  Sufficient  ether  (specific 
gravity  0-720)  should  be  used  to  fill  the  Soxhlet  tube  to  the 
top  of  the  siphon  one  and  a  half  times.  The  flask  is  sup- 
ported in  an  evaporating-basin  containing  water.  The 
basin  is  heated  by  a  small  flame.  The  ether  evaporates 
and  is  condensed,  running  back  over  the  paper,  soaking  it, 
and  filling  the  tube  until,  when  the  level  reaches  the  top  of 
the  siphon,  the  latter  acts  and  empties  the  whole  amount 
back  into  the  flask,  carrying  with  it  the  dissolved  fat. 
Twelve  such  siphonings  at  least  should  take  place,  and 
then  (the  flame  being  meanwhile  withdrawn),  the  condenser 
is  fixed  in  the  usual  position,  and  the  ether  distilled  over. 
The  flask  is  then  detached  and  dried  in  the  water-oven 
(laid  on  its  side)  to  a  constant  weight.  The  weight  obtained, 
less  the  weight  of  the  flask,  gives  the  weight  of  fat  in  5  c.c. 
of  milk  sample,  and  this  is  calculated  out  as  in  above 
example  to  a  percentage. 

Never  heat  the  flask  containing  ether  over  a  naked  flame, 
but  in  a  vessel  of  water  at  about  6o°  C,  keeping  the  ether 
in  a  gentle  state  of  ebullition. 

Gerber's  Process  is  somewhat  similar,  using  a  Gerber 
apparatus. 

Leffmann-Beam  Process. — Using  a  special  set  of  graduated 
bottles  and  a  special  centrifugal  machine.  Into  one  of 
the  bottles  15  c.c.  of  sample  are  introduced  by  means  of  a 
pipette,  and  then  3  c.c.  of  a  mixture  of  equal  parts  of  amylic 
alcohol  and  strong  HC1,  and  these  are  thoroughly  mixed. 
Then  9  c.c.  of  pure  concentrated  sulphuric  acid  are  added 
slowly,  1  c.c.  at  a  time,  shaking  after  each  addition.  The 
milk  will  gradually  assume  a  chocolate  colour  passing  on  to 
a  deep  brown.  Now  fill  up  bottle  to  zero  mark  with  a  hot 
and  freshly-made  mixture  of  one. part  of  sulphuric  acid  to 


76      PUBLIC  HEALTH  CHEMISTRY 

two  parts  of  water.  The  bottle  is  placed  in  the  machine, 
and  balanced  with  a  similar  one  filled  with  sulphuric  (i  in  2), 
and  the  machine  rotated  for  at  least  two  minutes.  On 
stopping,  the  fat  will  be  seen  to  have  separated  out  as  a 
layer  on  the  top,  and  the  percentage  is  read  off,  each  gradua- 
tion representing  o-i  per  cent  of  fat  by  weight.  The 
reading  is  from  the  extreme  top  of  the  fat  column  to 
extreme  bottom. 

There  is  also  a  Gerber  process  similar  in  principle. 

Maceration  Process. — Is  used  at  Somerset  House  in  the 
Government  Laboratory,  and  is  specially  suited  to  the 
analysis  of  sour  milks.  It  consists  in  neutralizing  the 
acidity  with  N  /io  strontia,  evaporating  to  the  consistency 
of  soft  cheese,  repeatedly  washing  with  ether,  which  is  run 
through  a  filter-paper  of  known  weight,  weighing  the  fat- 
free  solids  plus  the  filter-paper  washed  free  of  fat,  and  thus 
the  solids-non-fat  are  obtained,  an  allowance  being  made 
for  the  strontia  added.  This  figure  deducted  from  the 
total  solids  obtained  from  another  portion  of  the  sample 
gives  the  amount  of  fat.  (Several  corrections  are  made  in 
calculating  the  solids  for  alcohol,  for  volatile  acid,  and  for 
ammonia.) 

Calculation  Method  by  Hehner  and  Richmond's  Formula. — 
F  =  0-859  X  T  —  o-2i86  x  G,  where  F  represents  the 
percentage  of  fat,  T  the  percentage  of  total  solids,  and  G 
the  last  two  figures  of  the  specific  gravity  (including  any 
decimal).  For  skim  milks,  where  G  ~  T  exceeds  2-5,  the 
following  modified  formula  is  used  :  F  =  0-859  X  T  — 
0-2186  X  G  —  0-05  (G  -=-  T  —  2-5).  A  third  formula  gives 
T  where  G  and  F  are  known.  T  —  0-25  X  G  +  1-2  X 
F  -\-  0-14. 

Solids-not-Fat  —  Are  obtained  by  subtraction  of  the 
fat  from  the  percentage  of  total  solids.  Should  not  be  less 
than  8-5  per  cent. 

Calculation  of  Amounts  of  Adulteration  (by  Skim- 
ming and  Added  Water). — A  milk  may  be  skimmed 
of  fat,  or  have  water  added,  or  be  treated  in  both  ways. 
The  Board  of  Agriculture  has  fixed  the  standard  for  fat  in 
milk  at  not  less  than  3  per  cent,  for  solids-not-fat  not  less 
than  8-5  per  cent,  and  for  total  solids  not  less  than  11-5 
per  cent   (3  -f-  8-5).     Most  pure  milk  samples  give  total 


FOODS  77 

solids  =  12  per  cent  to  13  per  cent.  If  a  milk  sample 
gives  a  lower  figure  for  fat  or  solids-not-fat  than  these 
standards,  adulteration  is  assumed  to  have  been  practised, 
and  the  burden  of  proof  to  the  contrary  rests  with  the 
vendor  of  the  milk  sampled. 

1.  If  the  solids-not-fat  figure  is  less  than  8-5  per  cent, 
and  the  fat  figure  is  3  per  cent  or  above,  the  milk  has  been 
watered,  and  the  amount  of  water  added  is  calculated  from 

the  formula  : — (8-5—  SnF)  x  |^  =  percentage  of  added 

water. 

2.  If  the  fat  figure  is  less  than  3  per  cent,  and  the  SnF 
figure  is  8-5  per  cent  or  above,  then  fat  has  been  abstracted, 
usually  by  skimming,  or  already  skimmed  milk  has  been 
added.  The  percentage  of  fat  present  deducted  from  3 
per  cent,  when  multiplied  by  100  and  divided  by  3,  gives 
the  percentage  of  fat  abstracted.  (3  —  F)  X  *jp  =  per- 
centage fat  abstracted. 

3.  If  both  the  figures  (SnF  and  F)  are  below  the  standard, 
the  percentage  of  added  water  should  first  be  calculated,  and 
a  further  calculation  made  to  see  if  the  addition  of  this 
amount  of  water  would  account  for  the  lowness  of  the  fat 
figure.  If  it  does  not,  the  amount  of  fat  removed  is 
calculated  thus  : — The  percentage  of  fat  present  deducted 
from  that  found  by  calculation  after  allowing  for  the  water 
added  is  multiplied  by  100  and  divided  by  3.  Thus  a  milk 
having  7-65  per  cent  of  SnF  and  27  per  cent  of  F  would  be 
returned  as  having  10  per  cent  of  added  water.  If  the  SnF 
are  still  7-65  per  cent,  and  the  F  is  now  2-4  per  cent,  it 
would  be  said  to  have  10  per  cent  of  added  water  and  10 
per  cent  of  fat  abstracted. 

Lactose  in  Milk. — May  be  determined  by  the  Saccharo- 
meter  (certain  proteins  having  been  first  removed),  or  by 
Fehling's  test  as  now  described.  Take  10  c.c.  of  the  milk 
sample  in  a  test  tube,  add  a  few  drops  of  acetic  acid,  and 
warm.  The  casein  coagulates,  carrying  the  fat  with  it. 
Pour  into  a  Nessler  glass,  washing  out  all  the  curd,  and 
make  up  the  bulk  to  100  c.c.  Break  up  the  curd  and  filter 
several  times,  until  whey  is  as  clear  as  possible.  Fill  a 
burette  with  the  whey.  Take  10  c.c.  of  standard  Fehling's 
solution  in  a  porcelain  basin,  add  50  to  80  c.c.  of  aq.  dest., 


78  PUBLIC    HEALTH    CHEMISTRY 

and  bring  to  the  boil,  and  just  keep  boihng.  Now  add  the 
whey  from  the  burette  until  all  the  blue  colour  is  discharged.. 
The  end  reaction  is  difficult.  Allow  to  settle,  place  a  drop 
of  supernatant  liquid  on  a  white  tile,  add  a  drop  of  K  4FeCy  6, 
and  then  a  drop  of  acetic  acid.  A  brown  precipitate  shows 
that  some  copper  is  still  unreduced. 

Read  the  amount  of  whey  added,  divide  by  10.  This 
gives  the  amount  of  milk  which  exactly  reduces  10  ex.  of 
Fehling's  solution.  But  10  c.c.  of  Fehling's  solution  are 
reduced  by  0-0667  g1"111-  °f  lactose,  so  that  this  quantity 
of  milk  contains  0-0667  Srm-  °f  lactose,  and  the  amount 
in  100  c.c.  and  100  grm.  is  readily  calculated. 

Fehling's  Solution :  synonyms,  potassio-cupric  tartrate 
solution,  or  alkaline  cupric  tartrate  solution — consists  of 
34-65  grm.  of  crystallized  copper  sulphate,  CuS04*5H20, 
176  grm.  of  Rochelle  salt,  KNaC4H406'4H20,  77  grm. 
sodium  hydrate,  NaOH,  dissolved  in  water  and  bulk  made 
up  to  1000  c.c.  Of  this  solution  10  c.c.  are  completely 
reduced  by  0*05  grm.  of  glucose,  laevulose,  or  invert  sugar. 
Fehling's  solution  does  not  keep  well,  and  so  it  is  often 
made  up  in  two  parts,  equal  measures  of  which  produce, 
when  mixed,  Fehling's  solution.  Solution  No.  1:  34*64 
grm.  of  copper  sulphate  crystals  dissolved  in  water,  0-5  c.c. 
sulphuric  acid  added,  and  bulk  made  up  with  water  to  500 
c.c.  Solution  No.  2  :  176  grm.  Rochelle  salt  and  jj  grm. 
caustic  soda  dissolved  in  water,  and  bulk  made  up  to  500  c.c. 

Cane  Sugar  in  Milk. — (Present  in  preserved  milks, 
but  here  described  for  completeness.)  Take  a  portion  of 
the  whey  from  the  above  process,  and  boil  it  with  1  c.c. 
of  strong  HC1.  Cool  and  neutralize  with  anhydrous  sod. 
carb.,  and  make  up  bulk  originally  taken  to  three  times 
with  distilled  water.  Estimate  the  invert  sugar  produced 
as  for  lactose.  Subtract  the  percentage  of  lactose  pre- 
viously found  from  the  percentage  of  invert  sugar  now 
obtained,  and  the  remainder  is  the  amount  of  invert  sugar 
derived  from  cane  sugar.  This  multiplied  by  0-95  gives  the 
percentage  of  cane  sugar  present. 

Nitrogen  in  Milk — Is  the  most  constant  ingredient, 
and  never  falls  below  0-5  per  cent.  Ten  grams,  of  milk 
are  weighed  into  a  dry  Kjeldahl  flask.  Evaporate  to  dry- 
ness on  the  water  bath,  and  to  the  dried  residue  add  20  c.c. 


FOODS  79 

strong  sulphuric  and  5  grm.  pot.  sulphate.  Heat  gently 
over  a  Bunsen  flame  until  all  frothing  ceases,  and  then 
place  on  a  stand  over  a  small  Bunsen  flame  until  the  liquid 
is  colourless  and  quite  clear.  Cool  the  flask  and  contents, 
and  add  50  c.c.  of  water,  and  neutralize  with  strong  NaOH 
solution,  adding  slight  excess  to  make  alkaline.  Distil 
over  the  ammonia  into  20  c.c.  of  normal  acid  sulphuric,  the 
distillation  taking  about  an  hour.  Titrate  with  decinormal 
alkali,  using  methyl  orange  as  indicator,  and  the  difference 
in  the  titre  found  and  what  the  titre  should  be  represents 
the  amount  of  nitrogen  in  the  amount  of  milk  taken,  and 
this  is  converted  into  albuminoids  by  multiplying  by  6-39. 

Microscopic  Examination. — This  is  always  advisable. 
The  strictly  normal  constituents  are  round  oil  globules  of 
various  sizes  in  an  envelope  and  a  little  epithelium.  The 
abnormal  constituents  are  epithelium  in  large  amount,  pus, 
conglomerate  masses,  and  casts  of  the  lacteal  tubules.  The 
added  matters  may  be  starch  grains,  portions  of  seeds,  and 
chalk. 

Bacteriological  Examination. — See    Bacteriology. 

Proteids  in  Milk. — Ritthausen's  Method.  Consists  in 
precipitating  with  copper  hydrate,  which  carries  down  the 
fat  and  proteids.  The  precipitate  is  collected,  dried,  and 
weighed  on  the  filter  paper.  Deductions  are  made  for  the 
amounts  of  fat,  salts,  and  copper  hydrate  precipitate,  and 
the  weight  of  filter  paper,  and  the  remainder  is  the  weight 
of  proteids  in  the  quantity  of  milk  taken. 

Boiled  Milk. — To  10  c.c.  of  milk  sample  add  1  c.c.  of 
solution  of  ortol  and  a  few  drops  of  peroxide  of  hydro- 
gen solution.  In  unheated  milk  a  crushed-strawberry 
colour  is  produced.  If  the  milk  has  been  heated  above 
720  C.  (i8i-6°  F.)  no  colour  will  be  produced,  but  on  the 
addition  of  a  little  unheated  milk  the  colour  will  appear. 
As  milk  is  pasteurized  at  1590  F.,  such  milk  will  be  positive 
to  this  test.  Paraphenylenediamine  (replacing  ortol)  gives 
an  indigo-violet  in  milk  not  heated  above  780  C. 

Preservatives  in  Milk. — L.G.B.  Circular,  1906.  "  The 
presence  in  milk  of  formalin  to  an  amount  which  is  ascer- 
tained by  examination  within  three  days  of  collecting  the 
sample  to  exceed  1  part  in  40,000  (1  part  per  100,000  of 
formic   aldehyde)   raises   a  strong  presumption   that   the 


80      PUBLIC  HEALTH  CHEMISTRY 

article  has  been  rendered  injurious  to  health,  and  that  the 
purchaser  has  been  prejudiced  in  the  above  sense  ;  also 
that  similar  presumption  is  raised  where  boron  preserva- 
tives are  present  in  milk  to  an  amount  exceeding  57  parts 
per  100,000  or  40  grains  per  gallon."  A  Departmental 
Committee  of  the  Board  of  Agriculture  (1901)  recom- 
mended "  that  the  use  of  any  preservative  or  colouring 
matter  whatever  in  milk  offered  for  sale  in  the  United 
Kingdom  be  constituted  an  offence  under  the  Sale  of  Food 
and  Drugs  Acts,"  but  this  recommendation  has  not  yet 
received  the  force  of  law. 

The  commonest  preservatives  in  use  are  boric  acid  or 
borates  and  formic  aldehyde  (formaldehyde).  Sodium 
carbonate  is  sometimes  added  to  restrain  the  formation  of 
lactic  acid.  Salicylic  acid,  benzoates,  salt,  sulphites,  fluor- 
ides, boro-glycerin,  and  hydrogen  peroxide  have  all  been 
used  at  various  times. 

1.  Borax  and  Boric  Acid. — The  ash  of  the  milk  is  treated 
with  a  little  dilute  HC1,  so  that  it  is  distinctly  but  not 
strongly  acid,  and  a  piece  of  turmeric  paper  is  placed  in 
the  liquid,  and  the  dish  is  slightly  warmed  for  a  few  minutes. 
The  turmeric  paper  is  removed  and  dried  at  a  low  tempera- 
ture. If  even  so  small  a  quantity  as  o-oi  per  cent  (1  in 
10,000)  of  boric  acid  be  present  it  will  produce  on  the  paper 
a  reddish  colour  when  dry.  On  moistening  with  a  drop 
of  an  alkaline  solution  the  red  turns  a  greenish  black. 
Boric  acid  and  borates  are  the  only  substances  which  will 
give  this  change  of  colour  in  an  acid  solution. 

Richmond's  Test.  Take  equal  quantities  of  the  milk 
sample  in  two  test  tubes,  and  add  N/10  caustic  soda  to 
each,  drop  by  drop,  until  a  faint  pink  colour  appears.  To 
one  tube  add  an  equal  quantity  of  water,  and  to  the  other 
the  same  quantity  of  neutral  50  per  cent  solution  of  gly- 
cerin. If  boric  acid  be  present,  the  latter  tube  will  turn 
white,  while  in  its  absence  both  will  remain  the  same  colour. 

Another  test  is  to  take  part  of  ash  and  add  strong  sul- 
phuric acid  and  some  alcohol.  Put  in  a  dark  place  and 
light  the  alcohol,  when  in  the  presence  of  boric  acid  it 
burns  with  a  green  flame. 

2.  Formaldehyde. — (1)  Hehner's  test.  Take  5  c.c.  of  milk 
in  a  test  tube  and  add  as  much  water.     Some  90  per  cent 


FOODS  81 

commercial  sulphuric  acid  is  then  run  down  the  side  of  the 
test  tube,  so  that  it  forms  a  distinct  layer  at  the  bottom  of 
the  tube.  Pure  milk,  free  of  formaldehyde,  gives  a  greenish 
ring,  but  when  formaldehyde  is  present  a  violet  ring  is 
formed.  Pure  sulphuric  will  not  give  the  test,  but  will  do 
so  after  the  addition  of  a  few  drops  of  ferric  chloride.  The 
test,  therefore,  can  be  varied  thus.  To  the  dilute  milk  add 
the  ferric  chloride,  and  then  some  strong  sulphuric,  when 
the  milk  becomes  a  reddish-purple  colour  ;  but  carbolic 
and  salicylic  acids,  if  present,  would  give  confusion  colours. 
(Hehner's  test  reacts  with  I  part  in  200,000,  but  fails  with 
milks  containing  overo  -5  per  cent). 

(2)  Add  a  drop  of  carbolic  acid  to  the  dilute  milk,  and 
repeat  Hehner's  test,  using  pure  H2S04,  when  a  red  ring 
indicates  the  presence  of  formaldehyde. 

(3)  Jorissens  Test. — To  10  c.c.  of  milk  in  a  tube  are  added 
several  drops  of  a  10  per  cent  aqueous  solution  of  phloro- 
glucinol,  the  mixture  shaken,  and  a  few  drops  of  NaOH  or 
KOH  added.  Normal  milk  gives  no  reaction,  but  a  milk 
containing  as  little  as  1  part  of  formalin  in  20,000  gives  a 
fleshy-pink  coloration. 

(4)  Distil  100  c.c.  of  the  milk,  and  use  the  distillate  for 
these  tests. 

(a).  Repeat  test  number  (2)  above.    Detects  1  in  200,000. 

(b).  Schiff's  Magenta  Test. — Take  a  very  dilute  solution 
of  magenta  (fuchsin  or  nitrate  of  rosanilin),  and  decolorize 
with  sulphurous  acid,  adding  drop  by  drop.  Add  a  few 
c.c.  of  distillate,  and  watch  for  some  minutes  (or  set 
aside).  Traces  of  formaldehyde  bring  back  the  colour 
slowly.  The  test  is  also  said  to  be  got  in  the  filtrate  of 
curdled  milk. 

(c).  Tatten-Thonison  Test. — To  20  c.c.  of  distillate  add 
5  to  10  drops  of  a  reagent  made  by  adding  ammonia  to  a 
2.  per  cent  solution  of  silver  nitrate  until  the  precipitate 
first  formed  just  dissolves.  Set  aside  in  a  dark  place  for 
twenty-four  hours.  Darkening  almost  to  blackness  is  due 
to  formaldehyde,  but  a  slight  browning  may  be  disregarded 
(often  seen  in  silver  solutions). 

Formalin  is  a  40  per  cent  solution  of  formic  aldehyde 
(CH  20)  in  water.  Two  to  three  drops  keep  a  pint  of  milk 
fresh  for  3  to  4  days,  and  0-05  per  cent  preserves  milk  for 

6 


82  PUBLIC    HEALTH    CHEMISTRY 

months.  In  the  milk  trade  a  much  more  dilute  solution 
is  used,  namely  I  of  formalin  in  80  of  water  (2  oz.  per 
gallon)  equal  to  0-5  per  cent  of  CH  20.  Rideal  states  that 
one-fourth  of  a  pint  of  such  a  solution  in  17  to  18  gallons 
of  milk  (=  1  in  126  to  144)  keeps  the  milk  fresh  for  at  least 
three  days,  and  does  not  give  it  any  taste  or  smell.  This 
dilution  is  roughly  1  part  of  CH20  in  100,000. 

3.  Salicylic  Acid. — Now  rarely  used  in  this  country,  is 
still  largely  employed  on  the  Continent.  It  is  best  detected 
by  Pellet's  method  :  100  c.c.  of  milk  sample  are  diluted 
with  as  much  distilled  water,  and  heated  to  6o°  C. ;  1  c.c.  of 
acetic  acid,  and  some  mercuric  nitrate  are  then  added. 
The  resulting  curd  is  filtered  off  and  rejected.  The  filtrate 
is  repeatedly  extracted  with  ether,  the  various  extractions 
are  mixed,  a  portion  is  evaporated,  the  residue  dissolved  in 
distilled  water,  and  the  solution  tested  with  ferric  chloride 
solution.  A  violet  coloration  not  discharged  by  acetic 
acid,  is  positive.  When  present  in  considerable  amount, 
the  direct  addition  of  ferric  chloride  gives  a  pale  brown 
colour,  and  in  the  filtered  whey  a  violet  may  be  detected. 

4.  Benzoates. — Curdle  the  milk  with  acetic  acid,  extract 
the  whey  with  chloroform,  neutralize  carefully  after  dilu- 
tion. Add  ferric  chloride,  when  benzoates  give  a  buff- 
coloured  precipitate  insoluble  in  acetic  acid. 

5.  Sulphites. — 

(1)  Add  dilute  phosphoric  acid  and  heat  gently ;  observe 
odour  (SO  J . 

(2)  Take  20  c.c.  in  a  tube,  add  Zn  and  HC1.  Place  paper 
soaked  in  lead  acetate  solution  over  mouth  of  tube  and  heat 
latter  gently.  Darkening  indicates  presence  of  sulphites, 
but  a  negative  result  is  more  reliable. 

Hydrogen  Peroxide. — First  suggested  by  Budde  :  milk 
said  to  be  "  Buddeized."  To  10  c.c.  of  milk  sample,  add 
1  c.c.  of  1  per  cent,  ortol  solution  (freshly  made),  when  in 
presence  of  hydroxyl  a  dull  crimson  colour  is  produced 
unless  the  milk  has  been  heated  above  720  C,  when  the 
addition  of  a  little  fresh  milk  is  required.  Paraphenylene- 
diamine  similarly  gives  a  blue  coloration.  Schardinger's 
reagent  is  decolorized  by  normal  milk,  but  not  by  treated 
milk.     In  the  presence  of  organic  matter  hydroxyl  splits 


FOODS  83 

up  slowly  into  water  and  free  oxygen,  and  so  after  six  to 
eight  hours  will  not  be  found. 

Pasteurized  Milk. — By  this  term  is  meant  milk  which 
has  been  heated  to  a  temperature  sufficient  to  kill  the  less 
resistant  pathogenic  bacteria  without  altering  the  flavour. 
The  temperature  used  varies  from  yo°  to  850  C.  (1580  to 
i85°F.).  The  higher  temperature  has  been  advised  by 
Professor  Bang.  The  one  mostly  used  is  750  C.  (1670  F.)  for 
half  an  hour,  cooling  quickly  thereafter.  Over  90  per  cent 
of  the  organisms  are  killed,  but  the  sporing  forms  are 
not  destroyed.  Souring  of  pasteurized  milk  does  not  take 
place  owing  to  the  destruction  of  the  lactic  acid  bacilli. 
It  does  not  keep  for  more  than  three  days. 

Buddeized  Milk. — Fifteen  c.c.  of  a  3  per  cent  solution 
of  hydroxyl  are  added  per  litre  of  milk  (1  of  H  20  2  in  2222), 
and  the  mixture  heated  for  three  hours  at  510  C.  (123-8°  F.). 
Milk  so  treated  is  normal  in  taste,  and  keeps  fresh  for 
eight  days,  even  in  hot  weather. 

Homogenized  Milk. — Under  200  to  400  atmospheres' 
pressure,  milk  is  forced  through  very  small  openings,  and 
the  size  of  the  fat  globules  reduced  to  o-ooi  mm.  in 
diameter.  This  prevents  the  fat  from  rising.  Adams' 
process  gives  too  low  an  estimate  of  fat  in  such  milks. 

Dried  Milk. — By  passing  a  thin  layer  of  milk  between 
two  heated  rollers,  the  milk  is  immediately  desiccated  and 
reduced  to  a  fine  powder,  which  merely  requires  the  addi- 
tion of  water  to  bring  it  back  to  the  condition  of  ordinary 
milk.  The  temperature  of  the  rollers  is  no°  C.  (2300  F.). 
Such  milk  may  be  lacking  in  the  antiscorbutic  properties 
necessary  for  infants,  but  appears  to  possess  all  the  solids 
of  the  original  milk  in  a  sterile  form. 

Humanized  Cow's  Milk. — This  is  prepared  on  the  large 
scale  by  diluting  the  milk  with  an  equal  quantity  of  pure 
water,  and  subjecting  the  mixture  to  centrifugalization. 
This  divides  it  into  two  equal  parts,  one  of  which  contains 
practically  all  the  fat  of  the  original  milk,  but  only  half  of 
the  other  ingredients.  The  only  constituent,  therefore, 
notably  deficient,  will  be  the  sugar,  which  is  added  in  the 
proper  proportion.     The  amount  of  proteid  tends  to  be 


84  PUBLIC    HEALTH    CHEMISTRY 

too  low,  and  the  mineral  salts  too  high.     Paget's  Perfected 
Milk  Food  is  a  concentrated  humanized  milk  of  this  class. 

Dirt  in  Milk. — In  Dresden,  a  standard  of  not  more  than 
8  mgr.  per  litre  exists.  In  this  country  Dr.  Houston  has 
suggested  (1905)  two  standards  for  dirt  in  milk : 
(1)  Amount  of  filth  that  settles  on  standing  to  be  less  than 
100  mgr.  per  litre ;  (2)  This  apparent  filth  is  diluted 
with  water  and  put  in  centrifuge,  when  amount  of  deposit 
should  be  less  than  50  mgr.  per  litre.  Otherwise  expressed, 
(1)  is  10  parts  per  100,000,  and  (2)  is  5  parts. 


CREAM  :    CONDENSED    MILK  :     INFANTS'    FOODS. 

Cream. — The  composition  is  similar  to  that  of  milk, 
but  the  fat  is  much  higher  in  amount.  No  standard  is  laid 
down  in  this  country  ;  but  in  the  state  of  New  Hampshire, 
cream  must  contain  18  per  cent  of  fat.  Cream  is  at  times 
thickened  by  the  addition  of  such  agents  as  gelatin,  starch 
paste,  saccharate  of  lime  (called  viscogen),  and  condensed 
milk. 

Colouring  with  annatto  and  coal-tar  d^^es  is  known. 
Annatto  may  be  detected  by  adding  bicarbonate  of  sodium, 
and  then  immersing  a  strip  of  white  filter-paper  and  stand- 
ing overnight.  A  brown  stain  indicates  the  presence  of 
annatto.  Coal-tar  dyes  of  the  azo  group  give  a  pink  colour 
with  diluted  mineral  acids. 

Fat  is  estimated  by  the  Leffmann-Beam  process : 
2  grams,  of  cream  are  mixed  with  12  c.c.  of  water,  and  the 
mixture  is  poured  into  the  bottle,  the  remainder  of  the 
procedure  being  as  before  (page  75).  The  result  is  multi- 
plied by  777.  The  Werner-Schmidt  and  Adams'  processes 
may  also  be  used. 

The  Departmental  Committee  on  Preservatives  advised 
that  only  borax  and  boracic  acid  be  allowed  in  cream,  and 
in  an  amount  not  exceeding  0-25  per  cent,  expressed  as 
boric  acid  ;  and  the  amount  to  be  notified  by  a  label 
affixed. 

Cream  prepared  by  centrifugalizing  methods  contains 
45  to  65  per  cent  of  fat.  Devonshire,  Cornish,  or  clotted 
cream  is  prepared  by  warming  milk  in  pans  for  several 


FOODS  85 

hours.  The  cream  rises  in  a  more  coherent  layer  (50  to  60 
per  cent  of  fat). 

Condensed  Milk. —  Consists  of  unsweetened  milk  or 
sweetened  milk  or  sweetened  skimmed  milk,  concentrated 
by  evaporation,  usually  to  one-third  of  its  volume.  The 
addition  of  two  volumes  of  water  should,  therefore,  produce 
a  strength  equal  to  the  original. 

The  unsweetened  milks  are  well  prepared,  keep  well,  and 
contain  the  due  proportion  of  fat. 

The  sweetened  milks  form  the  largest  class,  and  for  the 
most  part  are  good.  The  dilutions  recommended  in 
nearly  every  case  produce  a  milk  very  much  below  the 
standard  of  ordinary  milk.  Such  milks  usually  contain 
rather  more  cane  sugar  than  milk  solids.  For  example  (a 
good  specimen)  :  Fat  11  per  cent,  proteins  10  per  cent, 
milk  sugar  14  per  cent,  ash  2-2  per  cent,  and  cane  sugar 
38  per  cent.  (Solids  not  cane  sugar,  11  +  10  -f-  14  =  35 
per  cent.) 

The  sweetened  skimmed  milks,  'or  separated  milks,  or 
machine-skimmed  milks,  are  very  inferior  to  the  above, 
containing  as  little  as  0-2  per  cent  of  fat,  and  generally 
about  only  1  per  cent.  Such  milk  is  unfit  for  the  sole 
nourishment  of  children ;  and  it  would  be  better  if  such  a 
statement  had  to  be  put  on  the  label. 

Analysis. — Mix  the  contents  of  the  tin  well,  weigh  out 
10  grm.,  dissolve  in  water,  and  make  up  the  bulk  to 
100  c.c.  Analyse  as  in  the  case  of  ordinary  milk,  except 
that  for  fat  the  Werner-Schmidt  method  is  inapplicable  in 
sweetened  milks,  owing  to  the  charring  of  the  cane  sugar. 
In  the  Adams'  process  use  carbon  tetrachloride  instead 
of  ether. 

Humanized  Condensed  Milk. — This  is  a  condensed 
milk  with  added  milk  sugar  and  cream,  but  no  cane  sugar. 
When  diluted  with  water  in  the  proper  proportion  it  is 
practically  identical  in  quantitative  composition  with 
human  milk. 

Infant  Foods. —  These  are  sold  as  a  substitute  for 
mother's  milk.  As  they  are  in  the  dried  state,  their 
composition  should  approximate  to  that  of  dried  mother's 
milk,  which,  according  to  Hutchison,  is  as  follows  : — 


86 


PUBLIC    HEALTH    CHEMISTRY 


Water 

Proteid 

Fat 

Carbo- 
hydrate 

Mineral 
matter 

Dried  mother's  milk    

diluted  i  :  7*5 

Fresh  mother's  milk    

Fresh  cow's  milk         

nil 

88-3 

877 

87-5 

122 
1-52 

1-62 

3'5 

26'4 
330 
3M 
40 

52-4 
6-55 
6-26 

4\3 

21 
026 
0-27 
07 

Most  of  these  substitutes  are  deficient  in  fat,  some  are 
deficient  in  proteid,  and  many  of  them  contain  unaltered 
starch.     They  may  be  divided  into  three  groups  : — 

1.  Cow's  milk  desiccated,  with  additions  or  alterations. 
These  only  require  the  addition  of  water.  Includes  Allen- 
bury  (Nos.  1  and  2),  Horlick's  malted  milk,  Carnrick's 
soluble  food,  Milo  food,  Manhu  infant  food,  and  Maltico. 
They  are  all  deficient  in  fat  and  too  rich  in  carbohydrate. 
The  first  three  and  the  last  contain  no  unaltered  starch. 

2.  Cereals,  usually  wheat,  of  which  the  starch  has  been 
partly  or  wholly  transformed  into  dextrins  or  malt  sugar. 
In  Mellin's  food,  Cheltine  maltose  food,  and  Hovis  babies' 
food  (No.  1),  all  the  carbohydrate  is  in  a  soluble  form  in  the 
powder,  and  there  is  no  starch  present.  In  Savory  & 
Moore's  food  and  Allenburys'  malted  food,  the  powder  con- 
sists of  wheat  flour  mixed  with  malt.  When  prepared 
according  to  the  directions,  most  but  not  all  of  the  starch 
is  converted  into  soluble  forms.  Benger's  food  is  a  mixture 
of  wheat  flour  and  pancreatic  extract,  and  in  it,  too,  most 
but  not  all  of  the  starch  is  converted  into  soluble  forms. 
The  proteid  is  also  partially  digested.  These  are  all  made 
with  milk,  or  milk  and  water. 

3.  Cereals,  wheat  flour,  oats,  and  barley,  with  the  starch 
unaltered.  To  some,  sugar  has  been  added.  Made  with 
milk,  or  milk  and  water.  Such  are  Ridge's  food,  Neave's 
food,  Frame  food,  Scott's  oat  flour,  Robinson's  patent 
barley  and  groats,  etc. 

Analysis. — Examine  for  starch  (microscope).  Total 
nitrogen,  by  Kjeldahl  X  57  =  proteid.  Phosphates,  sugar, 
starch,  dextrin,  and  cold  water  extract :  see  under  Cereals 
and  Wheat  Flour.  Fat,  estimate  by  Soxhlet,  or  with 
petroleum  ether  (see  under  Coffee). 


FOODS 


87 


Table  of  Infant  Foods  (Hutchison). 


Water 

Proteid 

Fat 

Carbo- 
hydrate 

Mineral 
matter 

Dried  mother's  milk 
Allenbury,  No.  I    .  . 
No.  2    .  . 
Horlick's  Malted  Milk 
Mellin's  Food 
Hovis  B.  F.,  No.  i  .  . 
Savory  &  Moore's 
Benger's  Food 
Frame  Food  Diet 
Scott's  Oat  Flour 

nil 

5'7 

3-9 

37 

6-3 

3'7 

4-5 

8-3 

5-o| 

5-8 

122 

9*7 
9-2 

13-8 
7-9 
7'7 

io-3 

IO-2 

13*4 
9'7 

26-4 
20-0 

15-0 
9-0 

trace 
0-20 
1-4 

1-2 
1-2 

5-o 

52-4 

60-85 

69-I 

70-8 

82-0 

86-6 

83-2 

79-5 

79-4 

78-2 

21 

3-75 
3-5o 
2-70 

3-8 

1-82 

o-6 

o-8 

i-o 

1-3 

BUTTER. 

Butter  is  the  fat  of  milk  clotted  together  by  shaking  or 
beating  at  a  low  temperature.  A  thin  bluish  fluid  separates, 
which  is  called  buttermilk.  Butter  consists  of  neutral  fats 
mixed  with  water,  a  small  amount  of  casein,  and  traces  of 
salts,  and  there  may  be  added  salt  (NaCl).  The  average 
composition  is  : — Fat,  78  to  94  per  cent ;  water,  8  to  12  per 
cent ;  curd,  1  to  3  per  cent ;  salt,  o  to  7  per  cent.  The  fats 
are  present  as  glycerides  of  certain  fatty  acids,  namely  : 
butyric,  caproic,  caprylic,  capric,  myristic,  palmitic, 
stearic,  and  oleic  acids.  The  first  four  are  soluble  in  hot 
water,  and  are  known  as  the  "  soluble  fatty  acids,"  the 
rest  as  the  "  insoluble  fatty  acids."  Bell's  analysis  of 
butter  fat  is  :  butyric  acid,  6-1  per  cent  ;  caproic,  caprylic, 
and  capric  acids,  2-1  per  cent ;  myristic,  palmitic,  and 
stearic  acids,  49-4  per  cent  ;  oleic  acid,  36-1  per  cent  ; 
glycerol  (calculated)  ,12-5  per  cent.  These  are  present  chiefly 
as  tributyrin,  tripalmitin,  and  triolein  (:  C  3H  5(0-C  4H  70)  3  : 
C3H6(O.C16H310)3:   C3H6(O.C18H330)3:). 

Adulterations. — The  important  ones  are  :  Addition 
of  water  in  excess,  the  substitution  of  foreign  fats,  salt  in 
excess,  starch,  boric  acid. 

Examination  of  Butter  for  Water,  Salt,  Curd, 

Fat,  and  Boric  Acid. 
The  fat  is  further  examined  by  the  Valenta  test,  for 
specific  gravity,  for  volatile  fatty  acids,  for  fixed  fatty 


88  PUBLIC    HEALTH    CHEMISTRY 

acids,  refractive  index,  iodine  absorption,  and  foreign 
oils.  Butter  may  also  be  examined  for  starch,  annatto,  or 
other  colourings,  benzoic  and  salicylic  acids,  and  with  the 
polariscope. 

Water.  —  Weigh  2  grin,  of  butter  into  a  weighed 
capsule,  and  dry  in  the  water-oven  to  constant  weight. 
The  drying  may  be  expedited  by  adding  1*5  c.c.  of  absolute 
alcohol  after  melting  the  butter  in  the  water-oven.  The 
amount  of  water  should  not  exceed  16  per  cent  ("  Butter 
Regulations,"  1902).  The  same  standard  now  applies  to 
margarine  ("  Butter  and  Margarine  Act,"  1907). 

Salt.— The  residue,  after  burning  off  the  fat  and  curd 
from  the  dried  butter,  may  for  all  practical  purposes  be 
taken  as  salt.  Or,  melt  2  grm.  of  butter  in  some  hot 
water,  make  up  the  bulk  to  200  c.c.  with  hot  water,  and  put 
into  a  separating  funnel.  Allow  fat  to  separate  to  top, 
take  20  c.c.  of  the  clear  liquid,  titrate  with  standard  silver 
nitrate  solution,  and  calculate  result  in  terms  of  NaCl.  It 
rarely  exceeds  10  per  cent.     Often  high  in  Irish  butter. 

Curd  or  Casein. — Varies  from  0-3  to  4  per  cent.  It  is 
usually  calculated  by  difference. 

Fat. — The  amount  of  fat  may  be  estimated  in  a  Soxhlet 
apparatus;  using  a  prepared  thimble  made  of  filter-paper ; 
or  by  washing  the  dried  solids  with  ether.  For  the  other 
processes  the  fat  is  separated  by  rilling  a  Nessler  glass  with 
butter,  and  melting  on  the  water-bath.  The  fat  gradually 
rises  to  the  top,  and  the  water  and  curd  sink,  so  that  three 
layers  are  noted ;  fat,  curd,  and  water.  The  fat  is  now 
filtered  through  a  dry  filter-paper  placed  in  a  funnel  sur- 
rounded with  hot  water.  Care  is  taken  not  to  pour  any 
of  the  wet  curd  or  water  on  to  the  paper.  The  water-free 
fat  is  then  used  for  the  various  processes. 

I.  Reichert-W ollny  Process  for  the  estimation  of  the 
volatile  fatty  acids. 

Five  grms.  of  the  liquid  fat  are  introduced  into  a  300  c.c. 
boiling-flask  ;  2  c.c.  of  50  per  cent  NaOH  solution  (free  from 
CO  2),  and  10  c.c.  of  92  per  cent  alcohol,  are  added.  The 
mixture  is  heated  for  fifteen  minutes  (under  a  reflux  con- 
denser) on  the  water-bath  kept  at  ioo°  C.  The  fats  are 
thus  saponified.     Remove  the  condenser,  and  continue  the 


FOODS  89 

heating  for  half  an  hour,  or  until  the  soap  is  dry.  This  is 
to  get  rid  of  the  alcohol.  Freshly  boiled  aq.  dest.  is 
added  cautiously,  while  still  hot,  to  the  amount  of  ioo  c.c, 
and  the  flask  carefully  heated  until  the  soap  is  dissolved. 
Thereafter,  40  c.c.  of  N/i  H2S04  are  added,  and  3  to  4 
pieces  of  pumice  stone,  and  the  flask  is  rapidly  connected 
to  a  condenser  by  a  tube  having  a  bulb.  Heat  at  first  with 
a  small  flame  to  melt  the  insoluble  fatty  acids  (liberated 
with  the  soluble  fatty  acids  by  the  action  of  the  sulphuric 
acid),  but  do  not  boil.  When  fusion  is  complete,  increase 
the  heat,  and  distil  over  no  c.c.  in  thirty  minutes.  Shake 
the  distillate,  filter  100  c.c,  add  0-5  c.c.  of  1  per  cent 
phenolphthalein  solution,  and  titrate  with  N/10  soda  or 
baryta.  The  number  of  c.c.  used  X  1*1  is  called  the 
Reichert-Wollny  number,  and  for  genuine  butter  varies 
from  24  to  32.  A  blank  experiment,  using  the  reagents 
alone,  supplies  a  correction  for  any  acidity  due  to  these. 

This  is  an  official  process,  recognized  by  the  Government 
Laboratory  and  the  Society  of  Public  Analysts.  Sundry 
details  are  given  in  the  official  description,  such  as  the 
length  of  neck  of  flask,  size  of  tubes  and  condenser,  etc., 
all  to  secure  uniformity  of  method  and  apparatus,  and  so 
give  comparable  results.  When  the  Reichert-Wollny  figure 
has  been  got,  if  it  is  24  or  above,  the  sample  is  looked 
upon  as  genuine,  and  as  1  c.c.  N/10  soda  =  0-0088  grm., 
or  8-8  mgr.  of  butyric  acid,  the  percentage  of  soluble  volatile 
fatty  acids  returned  as  butyric  acid  is  easily  calculated. 
With  the  Reichert-Wollny  varying  from  24  to  32,  the  per- 
centage of  acid  will  vary  from  4-2  per  cent  to  5-6  per  cent. 

Pure  Margarine  Fats  give  a  Reichert-Wollny  figure  of 
0-2  to  1  per  cent.  As  a  maximum  of  10  per  cent  of  butter- 
fat  is  allowed  to  be  added  for  flavouring  purposes,  the 
maximum  Reichert-Wollny  figure  for  a  saleable  margarine 
will  be  1  -f  10  per  cent  of  32  (=  3-2),  or  4-2.  The  figure 
usually  taken  is  3.  If  a  butter  gives  a  Reichert-Wollny 
of  say  y,  let  x  stand  for  the  percentage  of  margarine, 
then  3  x  x  +  24  (100-x)  =yx  100 ;  or  x  =  (2400  -  iooy) 
-T-  21. 

Unfortunately  for  the  value  of  this  process,  vegetable 
fats  which  give  a  moderate  Reichert-Wollny  figure  are  now 
being  used  for  food.      Thus  coco-nut  oil  has  a  Reichert- 


90  PUBLIC    HEALTH    CHEMISTRY 

Wollny  of  7,  palm-nut  fat  of  5,  and  certain  fish  oils  are 
said  to  give  values  above  40.  In  the  case  of  coco-nut  oil, 
the  figure  is  due  to  the  presence  of  the  glyceride  of  caprylic 
acid,  with  a  little  caproic,  but  no  butyric  glyceride.  To 
meet  this  difficulty,  several  tests  have  been  devised.  Here 
we  describe  that  of  Polenske,  which  is  the  most  generally 
adopted  one. 

Polenske  Method. — Is  based  on  a  determination  of  the 
insoluble  volatile  fatty  acids,  which  are  distilled  over  in 
the  Reichert-Wollny  process.  Certain  arbitrary  condi- 
tions must  be  observed  to  get  results  which  will  be  compar- 
able with  those  of  Polenske  and  others. 

Process. — Five  grm.  of  the  fat,  20  grm.  of  glycerol, 
and  2  c.c.  of  50  per  cent  NaOH  (an  alcoholic  solution  of 
soda  must  not  be  used)  are  taken  in  a  300  c.c.  boiling-flask, 
and  heated  over  a  naked  flame,  with  constant  shaking,  until 
a  clear  solution  is  obtained.  The  soap  formed  is  dissolved 
carefully  in  90  c.c.  of  hot  water  ;  50  c.c.  of  N/i  sulphuric 
acid  are  added  ;  and  o-i  grm.  of  pumice,  which  has  been 
powdered  and  then  sifted  through  muslin.  The  flask  is 
quickly  connected  to  the  top  of  an  upright  Liebig  con- 
denser, by  means  of  a  glass  tube  with  a  bulb  just  above 
the  cork,  and  the  tube  thereafter  bent,  first  at  an  obtuse, 
and  then  at  an  acute,  angle.  Distillation  is  commenced, 
and  the  flame  so  regulated  that  no  c.c.  are  distilled  over  in 
nineteen  to  twenty-one  minutes.  When  no  c.c.  have  been 
received  into  a  small  flask,  the  latter  is  replaced  by  a  25  c.c. 
cylinder,  and  the  flame  withdrawn.  The  cylinder  receives 
the  drainings.  The  flask  containing  the  no  c.c.  of 
distillate  is,  without  shaking  the  contents,  put  into  a 
bath  of  water  at  io°  C.  for  ten  minutes,  the  level  of  the 
water  outside  being  just  above  that  of  the  distillate  inside 
the  flask.  (At  this  stage,  it  may  be  noted  that  the  insoluble 
fatty  acids  are  almost  invariably  opaque  and  white  in  the 
case  of  butter,  while  10  per  cent  or  more  of  coco-nut  oil 
gives  clear  oily  globules.)  The  distillate  flask  is  now 
shaken,  and  the  contents  are  filtered.  The  Reichert  figure 
may  be  obtained  on  100  c.c.  of  the  filtrate.  The  filter- 
paper  is  kept ;  and  the  distillate  flask,  the  condenser,  and 
the  cylinder  are  washed  out  with  18  c.c.  of  distilled  water, 
which  is  then  poured  on  to  the  filter-paper.     This  paper 


FOODS  91 

now  contains  all  the  insoluble  volatile  fatty  acids  which 
have  distilled  over.  These  are  dissolved  in  alcohol,  and 
the  solution  is  titrated  with  N/io  baryta  +  phth.  The 
number  of  c.c.  required  is  called  the  "  new  butter  value  " 
of  the  fat.  This  varies  with  the  Reichert  figure,  being  1-3 
where  the  Reichert  is  20,  and  3-0  where  the  Reichert  is  30. 
An  increase  of  o-i  in  the  new  butter  value  corresponds  to 
an  addition  of  1  per  cent  of  coco-nut  oil.  The  mode  of 
calculation  is  thus  :  The  new  butter  value  of  *a  pure 
butter  having  the  same  Reichert  figure  is  subtracted 
from  the  new  butter  value  of  the  sample.  The  difference 
multiplied  by  10  gives  the  percentage  of  coco-nut  oil 
present.  Ten  per  cent  and  under  is  not  detectable  with 
certainty.  (See  article  on  "The  Estimation  of  Coco-nut 
Oil  in  Butter-fat,"  by  F.  W.  Harris,  F.I.C.,  in  the  Analyst, 
November,  1906.) 

Insoluble  or  Fixed  Fatty  Acids. — About  5  grm.  of  the 
fat  are  taken  in  a  flat  porcelain  dish  or  in  a  flask,  melted  on 
the  water-bath,  and  50  c.c.  of  methylated  spirit  or  absolute 
alcohol  added.  A  clear  yellow  solution  is  formed.  Now 
add  2  grm.  of  NaOH  or  KOH,  and  continue  the  heating, 
stirring  all  the  while.  The  fats  are  saponified.  In  five 
minutes  add  a  few  drops  of  water  ;  if  turbidity  ensues,  all 
the  fat  has  not  been  saponified  ;  continue  the  heating. 
Repeat  until  no  turbidity  ensues.  (If  too  much  water  is 
added,  some  of  the  fat  may  be  precipitated  from  solution. 
In  that  case  add  more  alcohol.)  Continue  to  heat  until  all 
the  alcohol  is  evaporated  and  a  jelly  of  soap  remains. 
Then  add  water,  almost  filling  the  dish,  and  in  this  the  soap 
dissolves.  Dilute  HC1  is  now  added  until  strongly  acid, 
and  the  fatty  acids  are  thus  liberated.  Heat  for  half  an 
hour  on  the  water-bath,  filter  through  a  weighed  filter- 
paper  (5  inches  diameter)  placed  in  a  hot-water  jacket, 
washing  all  the  fatty  acids  from  the  dish  with  repeated 
amounts  of  boiling  water,  until  the  filtrate  is  no  longer  acid. 
The  insoluble  fatty  acids  remain  on  the  filter-paper.  They 
are  solidified  by  putting  cold  water  in  the  jacket.  The 
paper  is  then  carefully  removed,  placed  in  a  small 
weighed  beaker,  and  dried  for  two  hours,  and  then  the 
beaker  and  contents  are  weighed.  The  percentage  of 
insoluble  fatty  acids  in  the  butter  fat  is  then  calculated, 


92      PUBLIC  HEALTH  CHEMISTRY 

and  ranges  from  86-3  to  88-5,  on  average  about  87-3.  All 
other  animal  fats  give  an  average  of  95-3. 

Valenta  Test. — Depends  on  the  intermiscibility  of  butter 
fat  and  strong  acetic  acid  at  a  low  temperature,  whereas 
animal  and  vegetable  fats  (except  coco-nut  oil)  do  not 
mix  until  a  much  higher  temperature  is  reached.  Glacial 
acetic  acid  (99  per  cent)  is  used.  Take  a  test  tube  and  add 
3  c.c.  of  the  fat  and  3  c.c.  of  the  acid.  Immerse  it  in  hot 
water  to  heat  the  contents,  which  are  stirred  all  the  while 
by  a  thermometer.  If  the  sample  is  pure  butter  fat,  it  will 
have  cleared  at  400  C,  and  it  is  then  allowed  to  cool,  still 
stirring,  and  the  temperature  noted  on  the  first  appearance 
of  turbidity.  For  butter  fat  this  should  be  from  320  to  360  C. 
Margarine  fat  will  not  clear  under  75  °  C.  An  abnormally 
low  Valenta  figure  suggests  the  presence  of  coco-nut  oil. 

Specific  Gravity  of  Butter  Fat. — Is  now  seldom  taken  at 
ordinary  temperatures,  it  being  more  convenient  to  take 
it  with  the  fat  in  a  molten  state,  at  ioo°  F.  The  fat  at 
1100  F.  is  poured  into  an  ordinary  specific-gravity  bottle, 
which  is  then  placed  in  water  at  ioo°  F.  The  stopper  is 
pushed  home,  and  the  bottle  dried,  cooled,  and  weighed. 
The  weight  of  the  same  bulk  of  water  at  ioo°  F.  is  similarly 
ascertained,  and  the  specific  gravity  calculated.  Pure  butter 
fat  ranges  from  9107  to  913-5,  but  mostly  between  911  and 
913.  Margarine  fat  ranges  from  901-5  to  906,  at  ioo°  F. 
The  specific  gravity  is  also  taken  at  ioo°  C.  by  the  Sprengel 
tube  or  the  Westphal  balance,  and  ranges  thus  :  Margarine, 
856  to  860 ;  butter,  865-3  to  866-8 ;  coco-nut  oil,  868  to 
872 — compared  with  water  at  15-5°  C. 

Refractive  Index. — Is  determined  on  a  special  instrument. 
At  350  C.  it  varies  for  butter  from  440  to  490,  generally  460. 
Margarine  gives  about  540,  and  coco-nut  oil  about  430. 
Its  use  is  less  valuable  now  than  formerly. 

Boric  Acid. — Is  detected  in  the  ash,  as  in  the  case  of 
milk.  In  order  to  prevent  loss  when  ashing,  a  few  drops  of 
milk-of-lime  solution  should  first  be  added.  To  estimate, 
take  25  grm.  of  butter  in  a  beaker,  add  25  c.c.  of  a  solution 
of  lactose  (6  per  cent)  and  sulphuric  acid  (4  c.c.  of  N/i 
per  cent).  Melt  in  water-oven,  mix  by  stirring,  allow  to 
settle,  and  pipette  off  20  c.c.  of  the  aqueous  portion. 
Titrate  at  ioo°  C.  plus  phth.  with  N/2  NaOH,  until  a  faint 


FOODS 


93 


pink  appears.  Then  add  12  c.c.  of  glycerol,  when  boric 
acid  is  liberated  and  pink  disappears.  Titrate  again  ; 
note  number  of  c.c.  used,  and  the  difference  X  0-0368 
gives  amount  of  boric  acid  in  20  c.c.  This  X  (100  +  per- 
centage of  water  in  butter)  4-20  =  percentage. 

Saponification  Equivalent  of  Koettstorfer  for  butter 
fat  varies  from  242  to  253,  and  for  margarine  fat  the  mean 
figure  is  about  284.  The  saponification  of  oils  by  alkalies, 
is  a  definite  reaction,  and  may  be  represented  by  the 
following  general  equation  where  F  stands  for  the  radicle 
of  the  fatty  acid :— C3H5  (OF)  3  +  3KOH  =  C3H5  (OH)  a 
+  3KOF.  Therefore,  if  we  know  exactly  the  amount  of 
alkali  necessary  to  saponify  the  oil  under  examination, 
we  can  to  some  extent  determine  the  nature  of  the  glycerides 
present.  The  amount  of  alkali  required  varies  with  the 
composition  of  the  fatty  acids  ;  the  lower  the  molecular 
weight,  the  higher  will  be  the  amount  of  soda  or  potash 
needed  to  saponify.  This  amount  may  be  stated  in  three 
ways  :  (1)  As  a  percentage,  that  is,  the  number  of  grammes 
of  alkali  absorbed  per  100  grm.  of  fat ;  (2)  The  number 
of  milligrammes  of  alkali  absorbed  per  1  grm.  of  fat.  This 
is  called  the  saponification  value,  and  the  figure  for  it  is 
ten  times  the  percentage,  since  it  is  parts  per  thousand  ; 
and  (3)  As  the  number  of  grammes  of  fat  which  would  be 
saponified  by  1  litre  of  normal  alkali ;  that  is,  in  the  case 
of  potash,  by  56  grm.  of  alkali.  This  is  called  the 
saponification  equivalent,  and  may  be  got  by  dividing  the 
percentage  of  alkali  absorbed  into  5600,  or  if  the  exact 
atomic  weights  are  used,  with  oxygen  ==  16  as  the  basis, 
into  5610.  The  following  table  from  Moor  &  Partridge 
summarizes  these  facts,  in  regard  to  the  chief  fats  : — 


Glyceride 

Formula 

Mol. 
Wt. 

KOH  abs.  % 

Sn  Value 

Sn 
Equiv. 

Butyrin 

C3H5(O.C4H70)3 

302 

55'7 

557 

IOO-67 

Laurin    .  . 

C3H5(O.C12H230)3 

638 

26-4 

264 

2I2-67 

Palmitin 

C3H5(OC16H310)3 

806 

20-9 

209 

268-67 

Stearin 

C3H5(OC1SH350)3 

890 

18-9 

189 

296-67 

Olein 

C3H5(O.C18H330)3 

884 

19-0 

190 

294-67 

Pure 
butter  fat 

\       composite 

— 

22-15 
to  23-3 

221 

to  233 

254  to 

240-7 

94      PUBLIC  HEALTH  CHEMISTRY 

Process.  —  Weigh  2  grm.  of  sample  into  a  200  c.c. 
flask,  add  25  c.c.,  approximately,  N/2  (seminormal) 
alcoholic  solution  of  KOH.  A  like  amount  of  KOH 
solution  is  run  into  an  empty  flask  for  a  blank  experiment. 
The  flasks  are  fitted  with  corks  carrying  vertical  tubes 
4  ft.  long  to  act  as  condensers.  Both  flasks  are  then 
heated  on  the  water-bath  for  not  less  than  thirty  minutes, 
with  frequent  agitation.  Thereafter  a  few  drops  of  phth. 
are  added  to  each  flask,  and  both  are  titrated  with  exactly 
N/2  HC1  solution,  1  c.c.  of  which  ==  0-02805  grm.  KOH; 
therefore  the  difference  between  the  two  titrations, 
multiplied  by  this  factor,  gives  the  amount  of  KOH  taken 
up  by  the  oil,  and  from  this  the  percentage  is  easily 
calculated,  and  the  Sn  value  and  the  Sn  equivalent.  The 
Sn  value  shows  the  number  of  milligrammes  of  KOH 
required  to  saponify  1  grm.  of  the  oil,  since  55-7  per  cent 
equals  557  per  1000,  or  557  mgr.  per  1  grm. 

Iodine  absorption  of  butter  fat  ranges  from  23  to  38  per 
cent,  and  for  margarine  from  40  to  55  per  cent,  but  is  not 
of  much  value  in  determining  the  amount  of  foreign  fat 
in  butter.  The  fats  of  the  oleic  series  readily  unite  with 
a  definite  quantity  of  I,  Br,  or  CI,  the  others  being 
indifferent. 

Sesame  Oil. — To  10  c.c.  of  fat  sample  add  10  c.c.  strong 
HC1  containing  o-i  grm.  of  cane  sugar.  Shake  thoroughly 
and  allow  to  stand,  when  in  the  presence  of  even  2  per  cent 
of  sesame  oil,  the  aqueous  liquid  becomes  crimson  coloured. 

Cottonseed  Oil. — Mix  equal  volumes  of  the  fat  and  a 
saturated  solution  of  Pb  acetate,  add  AmOH  and  stir 
quickly.  On  standing,  the  surface  layer  turns  an  orange- 
red  colour.. 

Starch. — Melt  sample  in  a  small  beaker  or  tube,  pour  off 
fat,  and  add  KI  solution  to  water  and  curd.  Normal 
butter  gives  a  reddish  coloration  only,  while  a  very  small 
trace  of  starch  will  give  a  blue.  Examine  curd 
microscopically. 

Polariscope. — Pure  butter  (using  gaslight)  on  rotation, 
so  that  the  two  Nicol  prisms  are  at  right  angles,  gives  the 
whole  field  equally  dark.  If  20  per  cent  or  over  of 
margarine  is  present,  it  is  impossible  to  darken  the  whole 
field,  no  matter  how  the  prisms  be  placed,  but  a  cloudy 


FOODS  95 

appearance  is  got.     No  selenite  plate  should  be  used,  and 
the  butter  should  not  have  been  melted. 

Annatto  and  other  Colouring  Matters. — If  the  colouring 
matter  of  a  butter  can  be  extracted  with  alcohol,  foreign 
colours  are  present,  as  the  natural  colouring  matter  is  not 
soluble  in  alcohol.  Annatto  may  be  detected  by  extraction 
with  methyl  alcohol  and  carbon  disulphide.  The  latter 
dissolves  the  fat  and  sinks,  the  alcohol  dissolves  the  colour 
and  floats.  Separate  alcohol  and  evaporate :  yellow  stain, 
which  turns  blue-green  on  adding  a  drop  of  strong  sulphuric. 
Turmeric,  saffron,  saffronette,  marigold,  and  the  azo  dyes 
are  also  used. 

Butter  v.  Margarine. — 

i.  Digest  fat  of  sample  with  alcoholic  solution  NaOH, 
when  butter  gives  pleasant  odour  of  butyric  ether  or 
pineapples,  and  pure  margarine  gives  a  tallow  smell. 

2.  Burn  a  small  piece  of  sample  on  platinum  foil  and 
extinguish  flame :  butter,  rather  pleasant  smell ; 
margarine,  tallow  smell. 

3.  Melting-point  of  fat.  Take  a  platinum  loopful,  dip 
in  water  alongside  thermometer,  and  heat  until  fat  just 
translucent :  butter  fat,  300  to.350  C.  ;  margarine  fat,  rarely 
above  280  C ;  coco-nut  oil,  20°-26°  C. 

4.  Valenta  test. 

*    CHEESE. 

Cheese  is  made  from  milk  by  the  action  of  rennet,  and 
consists  of  coagulated  casein  with  varying  proportions  of 
water,  fat,  and  salts.  It  may  be  made  from  whole  milk 
(Cheddar,  Cheshire,  Gloucester,  and  some  American 
cheeses),  from  skimmed  milk  (Dutch,  Parmesan,  Suffolk, 
and  Somerset  cheeses),  from  whole  milk  and  cream  (Stilton). 
Parmesan  is  made  from  goat's  milk  (partly  skimmed)  ; 
Roquefort  from  ewe's  milk  (partly  skimmed)  ;  and  Gorgon- 
zola  by  adding  in  the  process  of  manufacture,  powdered  bread 
crusts  on  which  moulds  have  been  allowed  to  grow.  Cream 
cheese  consists  of  the  fresh  curd  which  has  been  moderately 
pressed,  and  is  eaten  before  being  allowed  to  ripen. 
"  Ripening  "  of  cheese  is  supposed  to  be  a  decomposition 
whereby  the  casein  undergoes  a  fatty  change  with  the 


96  PUBLIC    HEALTH    CHEMISTRY 

formation  of  lime  salts  of  the  fatty  acids,  and  a  soluble 
compound  of  phosphoric  acid  with  the  casein.  Average 
composition  of  Cheddar  cheese  per  cent :  water  33-9, 
ash  4-2,  fat  30,  nitrogen  4-3,  casein  27-3.  Water  is  very 
high  in  cream  cheeses,  which  are  also  very  deficient  in  fat, 
in  nitrogen,  and  in  casein.  The  Reichert  figure  for  the  fat 
of  cheese  is,  from  its  similar  origin,  the  same  as  that  for 
butter  fat.  There  is  no  standard  for  cheese  in  this  country, 
and  a  cream  cheese  can  be  purchased  containing  less  fat 
than  a  milk  one,  unless  double  cream  be  asked  for.  A  good 
standard  would  be  not  less  than  30  per  cent  of  true  butter 
fat  and  no  starch  or  other  extraneous  matter  :  for  cream 
cheese,  a  40  per  cent  standard.  U.S.A.  standard :  50  per 
cent  milk  fat  in  dried  solids. 

Analysis.  —  Moisture  :  dry  2  to  5  grm.  of  sample  cut 
in  thin  slices,  to  constant  weight  in  water-oven  at  1050  C. 
(absolute  alcohol  hurries  drying). 

Ash. — Ignite  dried  cheese  at* as  low  a  temperature  as 
possible. 

Nitrogen. — Treat  1  grm.  of  sample  by  the  Kjeldahl 
process.      N  x  6-38  =  proteins. 

Fat. — Take  dried  cheese,  put  into  filter-paper  thimble, 
and  extract  in  Soxhlet  for  two  hours  ;  or  grind  50  grm. 
of  sample  in  a  mortar  with  sand,  put  mixture  in  tall 
stoppered  cylinder  and  extract  with  four  portions  of  ether, 
using  in  all  about  500  c.c.  Make  to  a  definite  volume, 
take  aliquot  part,  evaporate  to  dryness,  and  weigh  residue. 
Or  by  Leffman-Beam. 

Reichert-W ollny  Process.  —  Extract  fat  by  melting  in 
water-oven. 

Poisonous  Metals  in  Rind.  — Lead,  copper,  and  arsenic 
have  been  found  in  rind. 

Salt  5  to  6  per  cent ;  tyro-toxicon,  acarus  domesticus, 
moulds. 

CEREALS  I    WHEAT-FLOUR  I    BREAD. 

The  cereals  consist  of  the  edible  grains,  such  as  wheat, 
oats,  barley,  rye,  maize,  rice,  millet,  and  buckwheat. 
From  these,  various  products  are  got  which  are  largely 
used  for  food ;  as  wheat  flour,  oatmeal,  barley  meal,  pearl 
barley,  rye  flour,  corn  flour  (from  maize  or  Indian  corn), 


FOODS  97 

buckwheat  flour,  flaked  rice,  ground  rice,  rice  flour,  hominy 
(split  maize).  The  proteid  varies  in  the  different  cereals. 
Wheat  flour  has  the  largest  proportion  of  gluten,  and 
therefore  makes  the  best  bread.  Cane  sugar  is  found  in 
all  cereals. 

The  analysis  of  these  consists  in  estimating  the  moisture, 
ash,  fat,  phosphoric  acid,  total  nitrogen,  and  proteids  ; 
sugar,  starch  and  dextrin,  cellulose,  extract  and  acidity, 
and  true  albuminoids. 

Phosphoric  Acid. — Dissolve  ash  in  diluted  HC1,  boil  and 
filter.  Precipitate  lime  with  Am  2C  20 ,,  filter  after  heating 
on  water-bath  for  one  hour,  and  precipitate  phosphoric  acid 
from  filtrate  with  AmCl,  AmOH,  and  MgCl2.  Stand  for 
twelve  hours,  filter,  dry,  ignite,  and  weigh  as  magnesium 
pyrophosphate  Mg2P207. 

Sugar,  Starch  and  Dextrin. — Take  5  grm.,  and  boil  with 
250  c.c.  aqua  and  50  c.c.  N/i  HC1  under  an  invert  condenser 
for  six  hours.  If  hydrolysis  of  starch  is  complete,  a  drop 
of  the  solution  will  give  no  blue  colour  with  iodine  solution. 
Neutralize  with  50  c.c.  N/i  NaOH,  filter  into  a  litre  flask, 
and  with  washings  make  up  to  the  mark.  Estimate  with 
Fehling's  solution,  either  volumetrically  or  gravimetrically. 
The  Pavy-Fehling  method  is  more  reliable  than  the  ordinary 
way.  Pavy's  solution  is  made  from  Fehling's  thus : 
120  c.c.  of  Fehling  are  taken,  and  300  c.c.  strong  AmOH 
(sp.  gr.  o-88o)  and  400  c.c.  NaOH  solution  (12  per  cent) 
are  added,  and  the  volume  is  made  up  to  1  litre  ;  100  c.c. 
of  this  solution  has  the  same  oxidizing  effect  on  glucose  as 
10  c.c.  of  ordinary  Fehling  solution,  that  is,  100  c.c  ===  0-05 
grm.  glucose.  The  cuprous  oxide  is  not  precipitated  in 
the  presence  of  ammonia,  and  the  titration  is  continued 
until  the  solution  is  colourless.  In  working,  take  a  300  c.c. 
boiling-flask  fitted  with  a  two-holed  stopper.  Through  one 
hole  goes  the  nose  of  the  burette  containing  the  dilute  sugar 
solution,  and  through  the  other  a  tube  to  lead  away  the 
ammonia  ;  50  c.c.  of  the  Pavy  solution  are  taken  in  the 
flask  and  brought  to  the  boil.  The  sugar  solution  is  then 
run  in  slowly  from  the  burette  till  the  blue  colour  is  dis- 
charged.    To  convert  glucose  to  starch  X  0-9. 

Adulterations. — Talc,  gypsum,  French  chalk  ;  substitu- 
tion ;  blending ;  bleaching  with  NO  2  (also  O  3,  SO  2,  SO  3, 

7 


98  PUBLIC    HEALTH    CHEMISTRY 

CI,  etc.) ;  addition  of  "  improvers  "  (acid-phosphates  of  K, 
Mg  and  Ca;   H3P04;   diastase,  etc.). 

Animal  and  Vegetable  Parasites. — The  corn  weevil 
(Calandra  granaria),  the  ear  cockle  {Vibrio  tritici),  ergot 
(Claviceps  purpurea),  smut  (Uredo  segetum),  bunt  {JO redo 
fcetida  or  Tilletia  caries),  rust  (Puccinia  graminis),  Mucor, 
Aspergillus  and  Penicillium ;    Acarus  farince. 

Wheat  Flour. —  Examine  microscopically,  physically, 
and  practically  (by  making  bread).  Should  be  white  in 
colour,  silky  to  touch,  free  from  smell  or  odour,  and  not 
yellow  or  gritty.  Moisture  :  dry  5  grm.  in  water-oven 
(should  not  exceed  18  per  cent).  Ash:  ignite  (not  more 
than  1  per  cent).  Total  proteid,  from  Kjeldahl  N  x  6-25 
(not  less  than  8  per  cent).  Cold-water  extract,  (10  per  cent) 
(should  not  exceed  47  per  cent).     Acidity,  none. 

Glutin. — The  proteins  consist  chiefly  of  a  globulin  and 
an  albumose  which,  when  acted  on  by  water,  unite  to  form 
glutin  (from  gliadin  and  glutinin).  Estimate  thus  :  weigh 
10  grm.  of  flour  and  put  into  a  porcelain  dish.  Add  pure 
water  from  a  burette,  stirring  the  while  with  a  glass  rod 
so  as  to  make  a  well-mixed  dough.  Be  careful  to  add  the 
water  slowly,  using  about  4  c.c.  When  made,  let  the 
dough  stand  for  fifteen  minutes,  then  pour  a  little  water 
on  and  stir  about  with  the  rod  to  let  the  water  wash  out 
the  starch.  Repeat  until  washings  are  free  from  starch, 
as  tested  by  iodine.  (After  a  time  the  glutin  becomes  so 
coherent  that  it  can  be  worked  with  the  fingers.)  Dry,  and 
weigh.  If  time  does  not  permit  of  drying,  weigh  moist 
glutin  and  divide  weight  by  2-9.  Dry  glutin  ranges  from 
8  per  cent  to  12  per  cent  (reject  if  below  8  per  cent). 

Cold  Water  Extract. — Weigh  20  grm.  into  a  dry  flask, 
add  200  c.c.  aq.  dest.,  and  shake  for  five  minutes.  Allow 
to  stand  twenty-five  minutes.  Decant  off  clear  liquid  and 
filter  through  a  dry  paper.  Evaporate  50  c.c.  in  water- 
oven  to  constant  weight,  and  this  gives  total  soluble 
extract,  consisting  of  sugar,  gum,  dextrin,  soluble  albumin, 
and  phosphate  of  potash.  Ignite,  and  weigh  ash,  which 
is  phosphate  of  potash.  The  acidity  (if  any)  can  be 
determined  by  using  100  c.c.  of  the  cold-water  extract. 

Mineral  Matter. —  Shake  100  grm.  of  the  flour  with 
200  c.c.  of  chloroform  in  a  separator.     Allow  to  stand,  when 


FOODS  99 

the  flour  floats  to  the  top  and  any  mineral  matter  sinks  to 
the  bottom.  Draw  off,  dry,  weigh,  and  examine  micro- 
scopically. Any  soluble  matter  can  be  dissolved  and  tested 
for  alum,  while  the  insoluble  can  be  tested  for  CaS04, 
BaSO  4,  and  CaCO  3.  To  test  for  alum  :  add  NaOH,  white 
precipitate  solution  in  excess  of  NaOH,  but  re-precipitated 
on  adding  AmCl.  The  sulphuric  acid  present  in  alum  is  pre- 
cipitated by  barium  chloride  ;  precipitate  insoluble  in  HC1. 

Bleaching  of  Flour. — Chiefly  with  nitrogen  peroxide 
(N02),  produced  by  the  action  of  HN03  on  FeS04,  or 
combustion  of  NH3  in  air,  or  by  electric  sparks.  The 
NO  2  with  the  moisture  in  the  flour  forms  nitric  and  nitrous 
acids,  the  latter  of  which  is  readily  demonstrated  in  a  io 
per  cent  watery  extract  by  the  Ilosvay  method  (page  48) . 
(i  to  3  parts  as  N  per  million).  (See  Hamill  and  Monier- 
Williams,  Journal  of  Hygiene,  vol.  xi,  No.  2,  July,  191 1). 

Alum. — Make  10  grm.  of  flour  into  a  paste  with  10  c.c. 
of  water.  Add  1  c.c.  of  each  of  two  solutions,  namely, 
(1)  Fresh  tincture  of  logwood,  and  (2)  Saturated  solution 
of  ammonium  carbonate.  Mix  well  with  a  glass  rod.  If 
the  flour  is  free  from  alum,  the  mixture  is  a  pinkish  colour 
fading  to  a  dirty  brown.  If  alum  is  present,  the  pink 
changes  to  a  lavender  tint  or  even  to  a  blue.  In  such  a 
case  the  sample  should  be  put  in  the  water-oven  for  two 
hours  to  make  sure  that  the  colour  is  permanent. 

Adulterations. — Those  mentioned  under  cereals;  water; 
"  improvers  "  ;  also  foreign  seeds.  One  of  these,  the  purple 
cow- wheat  (Melampyrum  arvense),  is  not  injurious,  but 
gives  bread  from  the  flour  a  smoky- violet  or  bJuish-violet 
tint.  Two  others,  the  corn-cockle  (Agrostemma  githago) 
and  darnel  seeds  [Lolium  temulentum)  possess  toxic  powers. 
The  corn-cockle  is  detected  by  its  appearance  as  large 
black  seeds,  while  the  darnel  seeds  are  detected  by  the 
repulsive  taste  they  give  to  the  flour  and  the  greenish 
colour  produced  on  the  addition  of  alcohol  (pure  flour 
gives  a  straw-coloured  solution  with  a  more  or  less  agree- 
able taste).  Darnel  seeds  do  not  affect  the  colour  of  the 
bread,  but  produce  in  those  eating  it,  vertigo,  hallucina- 
tions, delirium,  and  narcosis. 

Bread  is  made  by  kneading  wheat  flour  with  water,  the 
coherence  of  the  dough  being  due  to  the  moist  glutin 


100  PUBLIC    HEALTH    CHEMISTRY 

formed.  The  porosity  of  bread,  which  is  essential  to  its 
easy  digestion,  is  produced  by  enclosing  in  the  dough 
minute  bubbles  of  carbonic  acid  gas.  This  is  accomplished 
in  one  of  three  ways,  viz.  : — 

1.  By  the  use  of  yeast  which  sets  up  fermentation  of  a 
small  portion  of  the  starch,  forming  alcohol  and  carbonic 
acid  gas. 

2.  By  the  use  of  baking-powders  containing  an  acid  salt 
and  a  bicarbonate,  which  on  being  moistened  givfc  off 
carbonic  acid  gas. 

3.  By  kneading  the  dough  with  water  charged  with 
carbonic  acid  gas  under  pressure  (Dauglish's  system), 
so-called  "  aerated  bread." 

The  "  germ,"  an  important  constituent  of  the  grain,  and 
rich  in  fat  and  proteid,  is  removed  in  modern  milling 
processes,  and  its  absence  makes  the  flour  whiter. 

Analysis. — In  sampling,  take  crumb  and  crust. 

Moisture. — Take  10  to  50  grm.  and  dry  to  constant 
weight  in  water-oven  (should  not  exceed  40  per  cent). 

Ash. — Take  10  grm.  of  bread,  moisten  with  strong 
solution  of  ammonium  nitrate,  dry,  and  carefully  ignite. 
If  ash  is  still  very  carbonaceous,  repeat,  and  a  clean  ash 
will  usually  be  obtained  (should  not  exceed  2  per  cent,  and 
part  insoluble  in  HC1  should  not  exceed  0-2  per  cent). 

Alum. — Take  10  grm.  of  bread  free  from  crust,  in  a  dish. 
Pour  over  them  100  c.c.  of  water  containing  5  c.c.  each 
of  the  logwood  and  ammonium  carbonate  solutions 
described  above.  Stand  for  five  minutes,  drain  off  excess 
of  liquid,  and  dry  in  dish  at  ioo°C.  Violet  or  blue  tint  if 
alum  is  present  ;  if  not,  a  brown  or  buff.  Detects  7  grains 
in  a  4-lb.  loaf.     Test  unreliable  when  bread  is  sour. 

Acidity. — Soak  10  grm.  of  bread  in  100  c.c.  of  aq.  dest. 
for  one  hour,  macerate,  and  then  titrate  with  N/10  NaOH 
and  return  result  in  terms  of  acetic  acid  :  1  c.c.  N/10 
NaOH  —  0-006  grm.  glacial  acetic  (part  of  the  acidity  is 
due  to  lactic  acid) .  Some  extract  with  hot  water,  or  digest 
on  water-bath  and  filter  a  portion.  Use  phenolphthalein 
as  indicator.  Should  be  less  than  0-115  Per  cent  or  8  gr. 
per  lb.     Alum  test  is  unreliable  when  the  bread  is  sour. 

Copper  Sulphate  has  been  detected  in  bread,  probably 
due  to  the  corn  having  been  steeped  in  a  copper  solution  to 


FOODS  1(/1 

prevent  a  growth  of  fungus.  It  is  detected  by  soaking  in  a 
solution  of  K  4FeCy  6,  acidulated  with  acetic,  when  a  purplish 
or  reddish-brown  coloration  indicates  the  presence  of  copper. 

Ergot  in  Flour.  —  (i)  Microscope  ;  (2)  Place  some 
flour  in  a  test  tube,  and  a  few  c.c.  10  per  cent  KOH,  and 
warm  gently  :  smell  of  propylamine  (like  warm  urinous 
napkin)  ;  (3)  Shake  up  2  grm.  of  flour  with  10  c.c.  of 
alcohol  (70  per  cent)  containing  1  /2  c.c.  HC1.  On  standing, 
a  red  colour  slowly  forms. 

Baking-Powders. — Definition :  "  A  baking-powder  may 
be  defined  as  a  salt  or  mixture  of  salts  with  or  without  a 
diluent  such  as  starch,  which  evolves  carbon  dioxide  when 
moistened,  and  on  heating  "  (Moor).  The  Sale  of  Food  and 
Drugs  Act,  1899,  enables  authorities  to  take  cognizance 
of  the  constitution  of  a  baking-powder  as  an  article  which 
"  ordinarily  enters  into  or  is  used  in  the  composition  or 
preparation  of  human  food." 

The  best  mixture  is  one  containing  the  proper  chemical 
proportions  of  good  cream  of  tartar  (KHC4H406)  and 
baking  soda  (NaHC03),  with  a  filling  of  about  20  per  cent 
of  pure  starch.  The  chemical  action  forms  rochelle  salt 
(KNaC4H406).  This  keeps  well,  reacts  slowly  and  in  a 
definite  way  ;  and  the  rochelle  salt  formed  has  a  very 
weak  retarding  action  on  ferments,  and  so  does  not  disturb 
the  digestive  processes. 

Alum  is  objectionable  from  inhibiting  the  digestive 
ferments,  precipitating  the  phosphoric  acid  and  phosphates, 
and  liberating  sulphate  of  soda,  which  is  purgative. 
Bisulphate  of  potash  also  liberates  the  last-named  salt. 

Tartaric  acid  and  acid  phosphate  of  lime  liberate  the  CO  2 
too  quickly. 

Ammonium  carbonate  and  superphosphate  of  lime  (free 
from  sulphate)  are  not  objectionable. 

Available  carbon  dioxide  should  be  at  least  8  per  cent 
by  weight. 
KHC4H406  +  NaHC03  =»  KNaC4H406  +  H20  +  C02. 

Numerous  prosecutions  for  the  presence  of  alum  have 
been  made. 

STARCHES. 

Take  a  clean  slide,  and  put  on  it  a  small  drop  of  water. 
With  a  platinum  loop,  transfer  a  portion  of  the  starch 


to2  PUBLIC    HEALTH    CHEMISTRY 

powder  to  the  water-drop,  and  mix.  Put  on  a  cover-glass, 
remove  excess  of  water,  and  examine  with  a  microscope, 
using  first  a  §  inch  objective  and  then  a  i  inch.  Observe 
carefully  the  following  points:  (i)  Shape ;  (2)  Size  ;  (3) 
Hilum  (presence  or  absence  of) ;  (4)  Striations  (presence  or 
absence  of).  Iodine  solution  shows  up  the  striae.  (1-1000). 
Starches  fall  into  five  groups  : — 

1.  Contour  round — the  wheat  group — wheat,  barley, 
and  rye.  These  all  have  grains  large  and  minute  (rye  has 
also  intermediary  sizes,  as  also  has  barley  to  a  less 
extent),  and  have  no  very  apparent  hilum  or  striae. 

2.  Contour  oval — pea,  bean,  and  lentil.  These  all  have 
grains  of  large  size  with  a  longitudinal  hilum,  with  very 
faint  striae. 

3.  Contour  ovoid — potato  and  arrowroot.  Large  granules, 
with  a  distinct  hilum  and  well-marked  concentric  rings  or 
striae.  The  hilum  is  at  the  smaller  end  (oyster  shape)  in 
potato,  and  at  the  other  end  in  arrowroot,  the  granules  of 
which  are  smaller  (except  tous-les-mois  variety). 

4.  Contour  semi-faceted — sago  and  tapioca.  These  have 
a  hilum  and  ill-defined  rings.  The  sago  grains  are  the  larger. 

5.  Contour  faceted — maize,  oats,  and  rice.  The  rice 
granules  are  the  smallest,  the  maize  the  largest,  and  they 
have  a  stellate  hilum. 

The  polariscope  gives  valuable  aid  in  differentiation. 

CARBOHYDRATES. 

Carbohydrates  are  compounds  of  carbon,  hydrogen,  and 
oxygen,  the  two  latter  being  present  in  the  same  proportion 
in  which  they  combine  to  form  water.  The  class  is  a  large 
one,  and  its  components  are  widely  distributed  in  Nature. 
The  carbohydrates  may  be  arranged  into  three  groups,  viz. : 

1.  Monosaccharids  or  Glucoses,  including  dextrose  (or 
grape  sugar),  laevulose  (or  fruit  sugar),  and  galactose. 

2.  Disaccharids  or  Sugars,  including  saccharose  (sucrose 
or  cane  sugar),  lactose  (or  milk  sugar),  and  maltose  (or  malt 
sugar). 

3.  Polysaccharids  or  Starches,  including  starch,  dex- 
trin, gum  and  cellulose,  inulin,  and  glycogen. 

From  allied  groups  certain  substances  used  in  bac- 
teriology are  derived,  and  are  enumerated  here  for  the 


FOODS  103 

sake  of  completeness.  These  are  raffinose  or  melitose 
(tri-saccharid),  arabinose  (pentose),  and  mannite,  dulcite, 
and  sorbite  (hexahydric  alcohols,  hence  better  called 
mannitol,  dulcitol,  and  sorbitol). 

The  carbohydrates  are  distinguished  from  one  another 
not  only  by  their  chemical  constitution  but  by  their  relative 
sweetness,  their  source,  their  power  of  crystallization, 
their  oxidation  products,  their  power  of  reducing  Fehling's 
solution,  their  action  on  polarized  light,  their  reaction  to 
the  yeast  and  other  ferments,  and  other  tests. 

Commercially,  sugar  is  obtained  from  the  sugar  cane 
(West  Indies),  beetroot  (Western  Europe),  the  maple  tree 
(Canada  and  U.S.A.),  various  palm  trees,  such  as  date  palm 
(India),  sago  palm  (Ceylon),  Palmyra  palm  (South  America 
and  Australia),  a  grass  plant  called  sorghum  (U.S.A.),  and 
maize  or  Indian  corn  (Mexico).  These  plants  all  yield 
the  Sugar  known  under  the  various  names  of  sucrose, 
saccharose,  cane  sugar,  beet  sugar,  etc.  Sugar  is  soluble  in 
i  part  of  its  weight  of  water  at  150  C,  in  J  part  in  cold 
water,  and  in  all  proportions  in  boiling  water.  It  dissolves 
with  difficulty  in  alcohol.  Its  sp.  gr.  is  1-606.  Its  aqueous 
solution  is  dextro-rotatory,  its  specific  rotatory  power  at 
200  C.  for  sodium  light  being  Ad  =  -f  66-5.  It  melts  at 
1600  C,  and  on  cooling  forms  an  amorphous  glassy  mass 
known  as  "  barley-sugar,"  which  in  time  loses  its  trans- 
parency and  becomes  crystalline.  At  1900  to  2000  C.  it 
changes  to  a  brown  non-crystallizable  mass  called  caramel, 
which  is  used  for  colouring  liquids.  It  does  not  reduce 
Fehling's  solution  nor  ferment  with  yeast.  When  boiled 
with  dilute  acids  it  is  decomposed  into  dextrose  and 
laevulose,  both  of  which  reduce  Fehling  and  ferment  with 
yeast,  but  rotate  the  plane  of  polarized  light  in  opposite 
directions.  The  lsevulose  has  the  greater  rotating  power, 
with  the  result  that  the  solution  has  now  a  lsevo-rotatory 
action,  and  is  hence  called  "  Invert  Sugar."  Cane  sugar 
forms  compounds  with  metals,  metallic  oxides,  and  salts, 
and  these  are  named  saccharates  or  sucrates,  such  as  sodium 
sucrate,  chloride  of  sodium  sucrate,  and  lime  sucrate. 
Cane  sugar  crystallizes  in  large  monoclinic  prisms.  Boiled 
with  nitric  acid  it  oxidizes  to  oxalic  and  saccharic  acids 
and  inactive  tartaric  acid. 


104  PUBLIC    HEALTH    CHEMISTRY 

Glucose  and  Lcevulose  are  found  in  equal  proportions  in 
all  sweet  fruits.  It  is  likely  that  cane  sugar  first  forms  in 
the  plants,  and  that  a  ferment  at  once  breaks  it  up  into 
grape  sugar  and  fruit  sugar,  the  mixture  forming  invert 
sugar.  The  saccharine  substance  which  bees  collect  and 
form  into  honey  is  a  mixture  of  these  two  sugars,  forming 
"  invert  sugar,"  and  hence  pure  honey  is  lsevo-rotatory. 

Glucose,  or  dextrose,  or  grape  sugar,  or  blood  sugar, 
reduces  Fehling's  solution,  ferments  with  yeast,  is  dextro- 
rotatory (-f  527),  and  forms  six-sided  crystals. 

Lcevulose,  or  fructose,  or  fruit  sugar,  closely  resembles 
glucose,  but  rotates  polarized  light  to  the  left  (—  95-5). 

Lactose,  or  sugar  of  milk,  occurs  in  the  milk  of  mammals. 
It  crystallizes  with  one  molecule  of  water  in  rhombic 
prisms,  has  a  faintly  sweet  taste,  is  sparingly  soluble  in 
water  (1  in  6  of  cold  and  1  in  2-5  of  hot)  and  is  insoluble  in 
alcohol.  It  reduces  Fehling's  solution,  ferments  readily 
with  lactic  ferment,  but  not  with  yeast  (or  very  slowly). 
With  dilute  acids  it  yields  galactose  and  dextrose.  Lactose 
is  dextro-rotatory  (+  527)  for  A(l. 

Galactose  is  got  from  lactose  by  heating  with  dilute 
sulphuric  acid.  It  reduces  Fehling,  ferments  with  yeast, 
and  is  dextro-rotatory  (-f  83-3  for  A,). 

Maltose  is  a  variety  of  sugar  formed  together  with 
dextrin  by  the  action  of  malt  diastase  upon  starch.  It 
can  also  be  produced  by  the  action  of  dilute  sulphuric  acid 
on  starch.  It  ferments  with  yeast,  reduces  Fehling,  is 
soluble  in  alcohol,  is  dextro-rotatory,  and  crystallizes  in 
white  needles  +  1  molecule  H  20.  In  the  sugaring  of 
starch  by  diastase  at  6o°  C,  two-thirds  maltose  and  one- 
third  of  dextrin  are  produced  thus  : — 

3C  6H  x  0O  5  -f  H  20  =  C  x  2H  2  20  x  x  -f  C  6H  x  0O  5 
Starch  Maltose  Dextrin 

Starch,  or  amylum,  is  found  in  the  cells  of  many  plants 
in  the  form  of  granules  of  varying  size  (0-002  m.m.  to 
0-185  mm.).  These  are  insoluble  in  cold  water  and  in 
alcohol.  When  heated  with  water  the  granules  swell  up 
at  500  C,  burst,  partially  dissolve,  and  form  starch  paste, 
which  turns  the  plane  of  polarization  to  the  right.  The 
soluble  portion  is  called  granulose;  the  insoluble,  starch 


FOODS  105 

cellulose.  Alcohol  precipitates  a  white  powder  (soluble 
starch)  from  the  aqueous  solution.  The  blue  coloration 
produced  by  iodine  is  characteristic  of  starch.  Heat 
discharges  the  coloration,  but  it  reappears  on  cooling. 
By  dilute  acids,  starch  is  converted  into  dextrin,  maltose, 
and  dextrose  ;  by  diastase,  into  maltose  and  dextrin,  as 
shown  above  ;   and  by  the  saliva,  into  dextrin  and  maltose. 

Dextrin  is  really  a  name  which  denotes  several  isomeric 
substances  usually  found  in  mixture  and  resembling  each 
other  very  closely.  They  form  gummy  amorphous  masses 
whose  aqueous  solutions  are  dextro-rotatory,  hence  the 
name  dextrin.  They  do  not  reduce  Fehling's  solution 
and  are  not  directly  fermented  by  yeast,  but  in  the  presence 
of  diastase,  the  dextrin  is  changed  to  dextrose  and  then 
fermented.  They  give  a  red  colour  with  iodine,  and  are 
much  used  as  substitutes  for  natural  gums. 

Inulin  is  a  polysaccharid  found  in  the  roots  of  dahlia,  in 
chicory,  and  in  many  compositae.  It  is  a  white  powder, 
soluble  in  boiling  water  to  a  clear  solution.  It  gives  a 
yellow  colour  with  iodine.  When  boiled  with  water  it  is 
completely  changed  to  fruit  sugar  (laevulose). 

Glycogen  occurs  in  the  liver  of  mammals.  It  is  a  mealy 
powder,  soluble  in  hot  H  20,  gives  a  reddish-brown  colour 
with  iodine ;  ferments  change  it  to  maltose,  and  dilute  acids 
to  dextrose.     It  is  precipitated  from  solution  by  alcohol. 

Raffinose,  or  Melitose,  is  a  trisaccharid  found  in  Australian 
manna,  in  the  flour  of  cotton  seeds,  in  small  amounts  in 
sugar  beets,  and  is  crystallized  from  the  molasses.  It  is 
very  soluble,  is  dextro-rotatory,  is  easily  fermented  with 
yeast,  but  does  not  reduce  Fehling's  solution. 

Arabinose  was  formerly  thought  to  be  a  glucose,  but  is 
really  a  pentose  ;  that  is,  contains  but  five  carbon  atoms. 
It  is  made  from  gum  arabic  by  boiling  with  dilute  sulphuric 
acid.  It  crystallizes  in  prisms,  is  slightly  soluble,  is  dextro- 
rotatory, reduces  Fehling's  solution,  but  does  not  ferment 
with  yeast.     Boiling  mineral  acids  convert  it  into  furfurol. 

Mannite,  or  Manniiol,  exists  in  three  states,  namely,  dex- 
tro,  laevo,  and  inactive.  It  has  a  sweet  taste  and  is  found  in 
44  manna,"  the  dried  sap  of  the  manna  ash  (Fraxinus  ornus). 
It  is  also  made  (with  difficulty)  by  the  action  of  nascent 
hydrogen  on  glucose.     It  oxidizes  to  saccharic  acid. 


106 


PUBLIC    HEALTH    CHEMISTRY 


Dulcite  is  another  of  the  hexahydric  alcohols,  and  is  found 
in  a  manna  from  Madagascar.  It  is  made  artificially  from 
lactose  or  galactose  by  treating  them  with  nascent 
hydrogen.     It  oxidizes  to  mucic  acid. 

Sorbite  occurs  in  mountain-ash  berries.  With  one 
molecule  of  water  it  forms  small  crystals  which  dissolve 
readily  in  water. 

Asparagine  is  the  monamide  of  aspartic  or  amidosuccinic 
acid.  It  occurs  in  many  plants  (asparagus,  beetroot,  peas, 
beans,  etc.).  It  may  be  crystallized  from  the  pressed 
juice  in  rhombic  prisms  +  iH  20.  It  ferments  in  the 
presence  of  albuminoids  to  ammonium  succinate.  There 
are  laevo  and  dextro  varieties. 

Inosite  (muscle-sugar)  is  a  crystallizable  substance,  with 
the  same  empirical  formula  as  glucose,  but  it  is  not  a 
carbohydrate  but  a  hexahydric  phenol  of  the  benzene 
series.     It  is  also  found  in  unripe  peas  and  beans. 


Table    of 

Chief    Characteristics    of    Aforementioned 

Substances. 

Name 

Formula 

Source 

Rotatory 

Fehling's 

Yeast 

Glucose    . . 

C6H1206 

Sweet  fruits 

Dextro 

+ 

X 

Laevulose 

Laevo 

+ 

X 

Galactose 

H 

Lactose 

Dextro 

+ 

X 

Saccharose 

Ci2**22^11 

Cane,  beet  . . 

- 

- 

Lactose    . . 

Milk 

+ 

- 

Maltose    . . 

M 

Starch 

+ 

X 

Raffinose . . 

*"*1  8**S2^16 

Molasses     . . 

- 

X 

Arabinose 

CsH.oO/6 

Gum  arabic 

+ 

- 

Dextrin    . . 

(C6H10O5)n 

Starch 

- 

- 

Starch 

Plant  cells  . . 

- 

- 

Inulin 

lf 

Chicory,  etc. 

Laevo 

- 

- 

Mannite   . . 

C6H8(OH)6 

Manna 

Dextro 

- 

- 

Dulcite     . . 

,, 

t 

- 

- 

Sorbite     . . 

t} 

Mountain  ash 

n 

- 

- 

Asparagine 

C4H8N2Os 

Asparagus  . . 

D  &  L 

Inosite 

C6H]2062H20 

Muscle 

+  Reduces. 


-   Not.         X   Ferments. 


Adonite,  C5H7  (OH)5  is  a  pentite  (from  Adonis  vernalis). 

Salicin  (C18H1807)  and  Coniferin  (C16H2208'2H20)  are 
glucosides. 

Analysis  of  Cane  Sugar. — Moisture,  0-5  per  cent  in 
refined  sugar,  to  6  per  cent  in  raw  sugar.     Ash,  from  a 


FOODS  107 

trace  to  2  per  cent.  Glucose,  not  more  than  o-i  per  cent  in 
refined,  or  2  per  cent  in  raw.  Saccharose  or  sucrose,  93  per 
cent  upwards.  Other  organic  matter.  Mineral  matter. 
Colouring  matter.     Sugar  mite  (Acarus  sacchari). 

Moisture. — Dry  5  grm.  in  air-oven  at  1050  C.  to  a 
constant  weight  (about  two  hours). 

Ash. — Treat  residue  with  pure  sulphuric  acid  and  ignite 
to  whiteness  (takes  twenty  minutes).  Nine-tenths  of  the 
sulphated  ash  are  taken  as  the  true  ash.  The  ash  of  sugar 
consists  of  salts  of  potash  and  soda,  lime,  alumina,  and  silica. 

Glucose. — Estimate  by  Fehling's  solution,  volumetrically 
or  gravimetrically. 

Saccharose,  or  Sucrose,  is  estimated  either  (1)  directly,  by 
the  polarimeter  (saccharimeter),  or  (2)  after  "  inversion," 
with  Fehling's  solution. 

1.  Polarization  is  employed  in  all  commercial  transactions, 
and  for  the  assessment  of  duty  by  the  Customs  authorities. 
It  depends  on  the  optical  property  of  some  bodies  to 
rotate  the  plane  of  polarized  light.  The  specific  rotatory 
power  of  an  optically  active  substance  is  the  amount  of 
angular  rotation  (in  degrees)  of  the  ray  of  polarized  light 
which  is  produced  when  a  solution  of  the  substance  con- 
taining 1  grm.  per  c.c.  is  examined  in  a  column  1  decimetre 
(100  m.m.)  long.  If  a  be  the  observed  angle  of  rotation 
in  the  sample,  p  the  weight  of  the  substance  per  100  c.c. 
of  solution,  /  the  length  of  the  tube  in  decimetres,  and  d 
the  sodium  light,  then  the  specific  rotatory  power  (A)d  is 
determined  from  the  formula — 

...  a  100  X  a        ,  100  X  a 

(A)«  -  r^jj^o  -  t^f       p  =  i  maj; 

To  determine  (A)d  for  pure  cane  sugar,  dissolve  10  grm. 
in  water  and  make  up  to  100  c.c.  Put  some  of  the  solu- 
tion in  a  polarimeter  tube,  examine,  and  substitute  in 
formula.  Now  examine  sample  similarly  and  compare 
results.  The  ratio  of  these,  multiplied  by  100,  gives  the 
percentage  of  pure  sugar.  Or,  in  the  formula,  simply 
substitute  already  ascertained  values  for  (A)d, — 

+  66-5  being  taken  for  saccharose, 
+  52-7  ,,         ,,        dextrose,  and 

—  95-5  „         „        laevulose 


108  PUBLIC    HEALTH    CHEMISTRY 

at  a  temperature  of  20°  C.  Clarify  dark-coloured  solutions 
with  basic  lead  acetate.  If  cloudy  only,  add  3  c.c.  cream 
of  alumina,  mix  well,  add  one  drop  of  PbAc,  shake,  and 
filter.  If  yellow,  repeat,  adding  more  PbAc.  If  brown 
or  black,  add  2  c.c.  of  10  per  cent  Na2S03,  then  PbAc 
solution  till  no  further  precipitate. 

2.  Estimation   by  Fettling' s  Solution The  sugar  must 

first  be  inverted  by  boiling  with  a  dilute  acid  and 
then  estimated  either  volumetrically  or  gravimetrically. 
(a)  Volumetrically.  Take  1  grm.  of  sample,  dissolve  in 
50  c.c.  aq.  dest.,  add  5  c.c.  concentrated  HC1,  and  heat  to 
yo°  C.  for  10  to  20  minutes  to  invert — 

C  x  2H  2  20 1  j  -j-  H  20  =  C  6H 1 20  6  -f  C  6H  j  20  6 
Saccharose         Dextrose  Laevulose 

Cool,  and  neutralize  with  NaOH,  make  up  to  100  c.c. 
(i.e.,  1  per  cent),  and  put  into  a  burette.  Take  10  c.c.  of 
Fehling's  solution  in  a  flask  or  porcelain  dish,  add  40  c.c. 
of  water,  and  bring  to  the  boil.  Run  in  the  dilute  sugar 
solution  carefully  until  all  the  copper  is  reduced,  keeping 
the  liquid  at  the  boiling-point  all  the  time.  The  end  point 
is  the  chief  difficulty  in  this  method.  Filtration  is  helpful, 
but  an  indicator  is  commonly  used.  Allow  to  settle, 
remove  a  drop  of  the  supernatant  fluid,  and  test  on  a 
white  tile  with  potassium  ferrocyanide  until  a  brown 
precipitate  is  no  longer  given  on  acidifying  with  acetic  acid. 
Or,  using  Harrison's  indicator,  take  several  drops  of  a 
freshly-made  mixture  (of  0-05  grm.  of  starch  boiled  with 
water,  10  grm.  of  KI  added,  and  the  bulk  made  up  to 
100  c.c.)  on  a  white  tile,  and  from  time  to  time  a  drop  of 
the  hot  liquid  (precipitate  and  all)  is  removed  and  placed 
on  a  spot  and  a  drop  of  acetic  acid  is  superimposed.  A 
blue  colour  develops  until  all  the  copper  is  reduced.  10  c.c. 
of  Fehling's  solution  =  0-0475  grm.  inverted  cane  sugar 
and  0-0500  grm.  dextrose,  laevulose,  or  invert  sugar,  and 
0-0678  grm.  lactose  and  0-0807  &rm-  maltose,  (b)  Gravi- 
metrically. Proceed  as  above  until  the  1  per  cent  inverted 
saccharine  solution  is  obtained.  Thereafter  take  20  c.c. 
to  50  c.c.  of  Fehling  and  boil.  Add  a  measured  quantity 
of  the  1  per  cent  solution  (but  always  less  than  will  com- 
pletely precipitate  the  Fehling  solution)  and  continue  the 


FOODS  109 

boiling  for  two  to  six  minutes,  when  there  should  be  a  red 
precipitate  and  the  liquid  should  still  be  blue.  Now  filter 
rapidly,  washing  the  precipitate  at  first  by  decantation 
and  then  on  the  filter-paper,  until  the  washings  contain 
no  copper.  Dry,  ignite,  and  weigh  as  CuO  (black  copper 
oxide).  Weight  X  0-4308  gives  cane  sugar,  and 
X  0-4535  gives  glucose.  The  CuO  is  hygroscopic  ;  therefore, 
for  accuracy,  at  least  two  ignitions  and  two  weighings  are 
necessary.  Also  prolonged  ignition  is  needed  to  oxidize  all 
the  Cu20  (the  filter-paper  having  a  reducing  action). 

Insoluble  Mineral  Matter. — Dissolve  a  considerable 
amount  in  water,  filter  on  to  weighed  paper,  dry,  and  weigh. 

Colouring  Matter. — Extract  with  alcohol  and  steep  a 
piece  of  wool  mordanted  with  aluminium  acetate.  Aniline 
dyes  are  dissolved  and  stain  the  wool.  The  natural 
colour  is  not  so  removed.  Ultramarine  is  used  to 
"  whiten  "  sugar,  as  also  is  methyl- violet,  both  correcting 
the  slight  yellow  tint  natural  to  sugar.  To  fix  the  natural 
yellow  tint,  chloride  of  tin  was  formerly  used,  but  not  now. 
Demerara  sugar  is  made  by  adding  sulphuric  acid  to  the 
massecuite  (the  finished  mass  of  sugar  crystals  and  syrup 
formed  by  concentrating  syrup  in  a  vacuum  pan)  so  as  to 
slightly  char  the  sugar  grains.  About  3  gallons  of  pure 
sulphuric  acid,  diluted  with  1-5  gallons  of  water,  are  used 
for  5  tons  of  sugar.  This  gives  the  bright  yellow  colour 
so  much  admired.  Animal  charcoal,  sulphurous  acid,  and 
peroxide  of  hydrogen  are  also  used  in  various  processes. 

The  sample  is  dyed  if,  when  treated  with  a  few  drops  of 
concentrated  HC1,  a  pink  colour  forms. 

Sugar  Mite,  or  Acarus  sacchari,  by  microscope. 

Sugar  of  Milk  or  Lactose  is  made  on  the  large  scale  from 
whey  or  skimmed  milk. 

Glucose,  or  Starch  Sugar,  is  made  on  the  large  scale  from 
the  starch  of  maize,  potato,  rice,  and  sago,  by  treating 
with  weak  (pure)  mineral  acid,  aided  by  boiling  under 
increased  pressure  until  some  of  the  liquid  added  to  alcohol 
gives  no  precipitate  of  dextrin  or  no  iodine  reaction.  The 
acid  is  then  carefully  neutralized  and  the  resulting  glucose 
solution  refined.  Poisoning  has  arisen  from  the  use  of 
impure  sulphuric  acid  in  the  process  (arsenic  in  beer). 


110  PUBLIC    HEALTH    CHEMISTRY 

GOLDEN    SYRUP  :     HONEY. 

Golden  Syrup. — When  the  syrups  no  longer  yield 
sugar  they  are  made  into  treacle.  Golden  syrup  or 
invert-sugar  syrup  is  also  largely  made  from  the  sap  of  the 
maple  in  Canada  and  U.S.A.,  thus  saving  the  time  and 
fuel  required  to  extract  the  sugar.  The  various  drainings 
from  the  crystallized  sugar  are  called  molasses.  This  passed 
through  a  charcoal  filter  becomes  bright  and  clear,  and, 
when  concentrated  to  the  required  viscosity,  forms  golden 
syrup.  In  the  case  of  maple  sap,  it  is  first  boiled,  and 
skimmed  and  strained  while  still  hot,  and  the  evaporation 
is  continued  to  a  density  of  1325,  or  equal  to  n  lb.  of  sugar 
to  the  gallon.  It  is  then  poured  while  hot  into  perfectly 
clean  pans  or  tins,  which  are  then  sealed  to  keep  out  the 
air.  Syrups  of  this  strength  will  not  granulate  under 
ordinary  conditions.  The  steps  of  the  process  can  be 
gauged  by  a  thermometer,  the  thin  sap  boiling  at  about 
2130  F.,  and  a  syrup  of  the  desired  strength  at  2350  to 
2400  F.,  giving  a  polarimeter  figure  of  over  8o°,  for  which 
a  bounty  was  at  one  time  paid  by  the  state  of  Vermont, 
U.S.A. 

Molasses  of  inferior  grades  is  used,  when  inverted,  for 
the  manufacture  of  alcohol,  for  feeding  cattle,  or  for  fuel. 
Sorghum  juice  is  also  a  source  of  syrup. 

The  chief  adulterant  of  golden  syrup  is  glucose,  which  is 
added  to  thicken  it  to  improve  the  appearance,  and  where 
the  syrup  contains  some  crystallizable  sugar,  to  prevent  its 
deposition.  The  presence  of  glucose  is  determined  by  the 
polarimeter,  the  specific  rotatory  power  of  genuine  golden 
syrup  being  taken  as  +  160,  and  of  glucose  syrup  as  +  no°, 
then  if  ad  be  the  observed  rotation,  the  percentage  of 
glucose  will  be 

ioo(ad-i6)  -7-  (110-16) 

The  reducing  power  of  golden  syrup  is  due  to  glucose  and 
invert  sugars  present.  A  second  estimation,  after  inverting 
any  cane  sugar  present,  shows  how  much,  if  any,  is  present. 
A  thirds  after  inverting  for  three  hours,  shows,  if  giving  a 
higher  result  than  the  second,  the  presence  of  maltose  and 
dextrin,  which  would  confirm  the  presence  of  glucose  or 
starch-derived  sugar. 


FOODS  111 

Honey  consists  of  the  saccharine  substance  collected  by 
bees  from  the  nectaries  of  flowers,  and  deposited  in  the  cells 
of  the  honeycomb.  Honeydew  is  a  secretion  of  the  leaves 
of  various  trees  and  plants,  and  is  also  gathered  by  bees. 

Honey  contains  dextrose  and  laevulose,  and  hence,  like 
invert  sugar,  is  laevo-rotatory  from  —  4  to  —  15,  and  the 
reading  is  not  altered  to  any  extent  by  inversion,  showing 
the  absence  of  cane  sugar.  If  the  reading  is  +,  it  indicates 
the  presence  of  glucose  or  cane  sugar,  and  if  after  inversion 
it  is  still  +,  then  the  substance  is  glucose.  If  the  reading 
is  +  and  very  high  (above  100),  the  presence  of  glucose 
is  almost  certain,  and  in  this  case  if  no  cane  sugar  is 
present  at  the  same  time,  inversion  will  not  change  the 
reading.  Cane  sugar  is  stated  to  be  present  in  natural 
honey  at  times,  up  to  8  to  10  per  cent,  and  so  a  reading 
of  +  2  may  be  passed. 

Honey  is  largely  adulterated  with  glucose,  starch  paste, 
malt  extract,  and  cane  sugar,  and  imitated  by  adding  a  piece 
of  genuine  honeycomb  to  a  jar  of  glucose  syrup.  On  micro- 
scopical examination,  genuine  honey  will  always  show  the 
presence  of  pollen  grains,  which  would  generally  be  absent 
from  a  filtered  honey.  (Mel  depuratum  B.P.  is  the  honey 
of  commerce  melted  in  a  water-bath,  and  strained  while  hot 
through  flannel  previously  moistened  with  warm  water.) 

Beeswax  melts  at  about  640  C.  (1470  F.)  and  is  carbonized 
by  strong  sulphuric  acid  on  boiling ;  paraffin  wax  melts  at 
540  to  570  C.  (1300  to  1350  F.)  and  is  not  carbonized  on 
boiling  with  strong  sulphuric  acid. 

Analysis. — Water  varies  from  15  per  cent  to  25  per  cent, 
and  is  estimated  by  dissolving  5  grm.  in  water,  making 
up  to  100  c.c,  and  drying  10  c.c.  mixed  with  10  grm.  of 
sand  at  960  C.  until  weight  constant.  Ash. — Ignite  2  to  5 
grm.  at  a  low  heat.  Varies  from  o-i  to  0-3  per  cent, 
and  is  alkaline.  If  more,  it  suggests  glucose ;  test  for 
CaS04,  and  neutrality. 

Polariscope  Reading. — Dissolve  the  "  normal  weight  "  of 
honey  in  water  and  make  up  to  100  c.c,  filter  through  a 
small  quantity  of  bone  charcoal  to  clarify,  and  examine  in 
saccharimeter. 

Glucose. — Syrup  ;  sticky  precipitate  with  one  part  of 
water  and  ten  of  methyl  alcohol. 


112  PUBLIC    HEALTH    CHEMISTRY 

MUSTARD. 

Mustard  is  the  seed  of  Sinapis  alba  and  S.  nigra.  It  is 
commonly  sold  as  a  powder.  Pure  mustard  contains  14 
per  cent  of  carbohydrates,  o-66  per  cent  of  volatile  oil,  35 
per  cent  of  fixed  oil.  The  chief  adulterations  are  :  the 
addition  of  starch,  bringing  up  the  percentage  of  carbo- 
hydrates to  67  per  cent ;  the  abstraction  of  oil,  reducing 
it  to  as  low  as  7  per  cent  ;  the  addition  of  turmeric  to 
colour,  and  cayenne  pepper  to  make  the  taste  sharper. 
Examine  with  microscope  for  hexagonal  cells  in  white 
mustard. 

PEPPER. 

Black  pepper  is  derived  from  Piper  nigrum.  White 
pepper  is  made  from  the  inside  of  the  berry. 

Moisture,  9  to  11  per  cent ;  ash,  2  to  5  per  cent  ;  ash 
insoluble  in  HC1,  under  1  per  cent  for  white,  under  2  per 
cent  for  black  pepper  ;  fibre,  4  to  6  per  cent  in  white,  8  to 
11  per  cent  in  black  ;  carbohydrate,  65  per  cent  in  white, 
50  per  cent  in  black  ;  piperin  and  fixed  oil,  8-2  per  cent 
in  white,  7-8  per  cent  in  black.  Adulterations  :  linseed, 
mustard  husks,  wheat  and  pea  flour,  rape  cake,  ground 
rice,  ground  olive  stones  (poivrette),  sweepings. 

GINGER. 

Ash,  under  3-9  per  cent ;  soluble  ash,  over  1-7  per  cent  ; 
cold  water  extract,  over  8-7  per  cent. 

PEAS. 

Test  peas,  especially  when  tinned  or  bottled,  for  copper, 
used  in  "  greening  "  them. 

Qualitative  Test. — Acidify  with  HC1  and  put  a  piece  of 
bright  steel  in  the  liquid  in  which  the  peas  are  immersed. 
Stand  all  night.     Deposit  of  copper,  of  a  coppery  colour. 

Quantitative  Test. — Ash  20  grm.  of  peas,  boil  ash  with 
dilute  sulphuric  acid.  Filter,  and  make  up  filtrate  to 
50  c.c.  Test  amount  of  copper  colorimetrically,  or 
gravimetrically. 

MEAT  EXTRACTS  AND  ESSENCES. 

Total  nitrogen,  by  Kjeldahl ;  protein,  N  x  6-25 ;  moisture  ; 
ash  ;  reaction  ;  carbohydrate  ;  fat ;  antiseptics  ;  poisonous 
metals. 


CHAPTER      VI. 

BEVERAGES. 

COFFEE. 

The  seed  or  berry  of  the  plant  Coffea  arabica.  Each 
coffee  bean  contains  two  seeds.  These  are  removed, 
roasted,  and  ground,  producing  coffee.  The  chief  con- 
stituent is  caffeine,  identical  with  theine,  or  trimethyl- 
xanthin  C5H(CH3)  3N402,  and  on  the  average  1*2  per 
cent  is  present. 

The  chief  adulterant  is  chicory,  or  the  wild  endive 
(Cichorium  intybus),  but  this  can  be  legally  used  as  a 
diluent  for  coffee  if  the  article  is  sold  as  a  mixture.  Chicory 
is  the  root  of  the  plant,  dried,  and  powdered.  It  contains 
no  caffeine,  much  less  fat  than  coffee,  and  much  more  sugar. 

Other  adulterants  which  have  been  used  are :  dandelion 
root,  mangel-wurzel,  turnips,  bean,  pea,  rye,  and  wheat 
flours,  caramelized  condemned  sea-biscuit.  The  berries 
themselves  are  sometimes  spurious,  being  moulded  from  a 
composition  of  chicory  and  other  substances.  Chicory 
itself  is  sometimes  adulterated  with  some  of  these  substances 
and  occasionally  with  roasted  beetroot. 

Analysis. — 

Moisture. — Dry  5  grm.  to  a  constant  weight  at  ioo°  C. 
Should  not  exceed  6  per  cent  (chicory  10  per  cent). 

Total  Ash. — Ignite  5  grams,  until  a  nearly  white  ash  is 
obtained.  From  3-5  to  5  per  cent.  Chicory  ash  is  reddish 
from  the  presence  of  iron,  and  is  about  5  per  cent.  Four- 
fifths  of  coffee  ash  is  soluble  in  water  ;  one-third  of  chicory 
ash. 

Caffeine  is  extracted  by  successive  boilings  with  water, 
the  albuminous  matter  precipitated  by  acetate  of  lead, 
filter,  concentrate  the  filtrate  to  small  bulk,  and  extract 
four  or  five  times  with  chloroform.  On  evaporating  the 
chloroform,  pure  caffeine  is  left.  Should  be  ri  per  cent 
to  1 -3  per  cent  (chicory  none).  ^ 

8 


114  PUBLIC    HEALTH    CHEMISTRY 

Fat. — Soak  in  petroleum  ether  for  several  hours,  pipette 
off  20  c.c.  of  ether  and  evaporate  off  ether  in  a  tarred  vessel. 
Averages  n  to  14  per  cent  (chicory  1  to  2  per  cent). 

Sugar. — Extract  with  hot  water,  invert,  and  estimate. 
Under  1  per  cent  (chicory  10  to  18  per  cent). 

Starch. — Make  a  10  per  cent  decoction,  boil  with  animal 
charcoal  to  decolorize  (or  acid  permanganate),  cool,  and 
test  with  iodine  solution.  Pure  coffee  contains  almost  no 
starch,  and  the  presence  would  indicate  adulteration  with 
breadcrumbs,  etc.  Mounting  in  iodine  (1  in  1000)  brings 
out  the  concentric  rings. 

Detection    of  Chicory. — 

Qualitative. — Throw  a  pinch  on  to  the  surface  of  a  glass 
of  water.  Pure  coffee  remains  floating  on  the  top  for 
some  minutes  at  least,  whilst  chicory  sinks  almost  instantly, 
and  colours  the  liquid.  The  fragments  which  fall  to  the 
bottom  are  taken  out  separately  and  examined  by  the 
fingers  (coffee  is  hard,  chicory  is  soft).  They  are  then 
examined  microscopically  for  the  long  testa  cells  of  coffee 
berry  and  the  ladder-shaped  structures,  and  in  chicory  for 
the  cells  and  dotted  ducts. 

Quantitative. — Take  10  grm.  of  sample  in  a  boiling-flask, 
add  100  c.c.  of  water,  bring  to  the  boil,  and  continue  for 
one  half  minute.  Strain  through  muslin  or  a  fine  sieve, 
and  then  through  a  filter-paper.  Cool  to  15-5°  C.  (6o°  F.) 
and  take  specific  gravity  by  bottle.  Under  these  con- 
ditions pure  coffee  gives  a  decoction  having  a  sp.  gr.  rarely 
exceeding  1009-5,  and  chicory  about  1024  to  1025,  or  say 
1024-5.  Thus  a  difference  of  15  per  1000  represents  the 
difference  between  100  per  cent  coffee  and  100  per  cent 
chicory,  and  from  these  data  the  amount  of  adulteration 
can  be  calculated.  Say  that  the  sp.  gr.  of  sample  is  1014-5, 
that  is  a  rise  of  5  ;  therefore,  as  15  :  5  :  :  100  :  33-3  per  cent 
of  chicory,  and  a  fall  of  10  from  the  sp.  gr.  of  chicory,  and 
therefore  as  15  :  10  :  :  100  :  66-6  per  cent  of  coffee. 

A  second  method  is  to  take  10  grm.,  add  100  c.c,  bring 
to  the  boil,  filter,  boil  dregs  with  another  150  c.c.  for  five 
minutes,  allow  to  settle,  decant  off  clear  liquid,  mix  with 
previous  filtrate,  make  up  to  250  c.c.  with  aq.  dest.,  mix 
thoroughly,  pipette  off  50  c.c,  evaporate  to  dryness  in 
weighed  capsule  over   water-bath,    weigh,   and   calculate 


BEVERAGES  115 

as  a  percentage.  Coffee  gives  a  remarkably  constant 
percentage  of  24,  and  chicory  a  mean  percentage  of  70,  or 
a  standard  difference  of  46,  so  that  percentage  of  coffee  = 
100  (70— percentage  of  extract  found)  -1-  46.  A  third 
method  is  to  calculate  the  percentage  of  soluble  ash  and 
then  calculate  adulteration  on  this  basis. 


TEA. 

Tea  is  the  prepared  leaf  of  the  shrub,  Camellia  thea, 
which  grows  in  China,  India,  Ceylon,  and  Japan.  It  is 
nearly  always  blended  to  a  standard  quality  and  flavour 
by  mixing  two  or  more  kinds  of  leaves.  When  the  leaves 
are  baked  (to  dry  them)  immediately  after  picking,  green 
tea  results  ;  but  if  first  allowed  to  ferment  and  then  baked, 
black  tea  is  obtained.  The  chief  constituents  of  tea  are 
moisture,  caffeine,  tannin,  albuminous  matters,  ethereal 
oil,  gum,  dextrin,  fat,  wax,  chlorophyll,  woody  fibre,  etc. 
Tea  is  now  rarely  adulterated,  since  it  is  examined  by  the 
Customs  authorities,  and  samples  found  to  be  adulterated 
are  not  allowed  to  be  imported.  The  usual  adulterants 
were  foreign  leaves  (sloe,  willow,  elder,  hawthorn,  beech, 
etc.),  sweepings,  tea  dust,  clay,  mineral  matter,  ground 
olive  stones,  catechu,  gum,  starch,  and  exhausted  tea- 
leaves. 

Moisture. — Dry  5  grm.  of  sample  to  a  constant  weight. 
Varies  from  4  to  11  per  cent  ;    average  6  per  cent. 

Total  Ash. — Ignite  dried  tea  at  lowest  possible  tempera- 
ture to  get  a  grey,  not  a  green  ash.  Varies  from  about 
5  per  cent  to  7  per  cent,  averaging  about  6  per  cent. 

Insoluble  Ash. — The  total  ash  after  weighing  is  washed 
on  to  a  filter-paper,  and  thoroughly  extracted  with  about 
400  c.c.  of  very  hot  water,  the  washings  being  carefully 
preserved.  The  filter-paper  is  then  dried,  ignited,  and 
weighed,  and  the  weight  of  filter-paper  ash  being  deducted, 
the  amount  of  insoluble  ash  is  known.  Varies  from  2  per 
cent  to  4  per  cent,  of  which  less  than  1  per  cent  is 
insoluble  in  HC1. 

Soluble  Ash. — Deduct  amount  of  insoluble  ash  from 
total  ash,  and  result  is  soluble  ash.  Varies  from  2-8  to  4 
per  cent,  but  should  be  at  least  half  the  total  ash.     If  the 


116  PUBLIC    HEALTH    CHEMISTRY 

soluble  is  low,  the  inference  is  an  admixture  with  exhausted 
leaves. 

Alkalinity  of  the  Soluble  Ash  is  determined  by  titrating  the 
washings  of  the  total  ash  (taking  50  c.c.)  with  N/10,  HC1  or 
H2S04,  using  methyl-orange  as  indicator  and  returning 
the  answer  in  terms  of  K20.  Varies  from  1-3  to  2  per 
cent.    1  c.c.  N/10  acid  =  0-0047  grm-  K20. 

Extract. — Dry  some  of  the  leaves  at  ioo°  C. ;  then 
weigh  out  2  grm.  and  exhaust  thoroughly  by  boiling  under 
a  reflux  condenser  for  one  hour.  Filter  off  water,  and  repeat 
with  more  water  until  no  more  colour  is  imparted  to  the 
water.  Collect  the  exhausted  leaves,  dry,  and  weigh.  The 
difference  from  original  weight  gives  the  amount  of  extract, 
which  should  be  from  35  to  40  per  cent  of  the  dried  tea. 

Caffeine. — Extracted  as  indicated  under  "  Coffee." 
Varies  from  i-8  to  3-5  per  cent ;  average  2-6  per  cent. 

Tannin. — Treat  2  grm.  as  described  under  "  Extract/' 
but  keep  the  filtrates  and  make  up  to  a  known  bulk  (1  litre). 
Take  an  aliquot  part  (say  100  c.c.)  in  a  beaker,  add  excess 
of  5  per  cent  copper-acetate  solution,  and  boil.  The 
precipitate  contains  all  the  tannin.  Filter  and  wash 
precipitate  until  the  washings  are  free  of  copper.  Dry, 
ignite,  weigh,  and  deduct  filter  ash.  The  weight  of  ash  x 
1-305  =  tannin.  The  ash  is  CuO.  The  amount  of  tannin 
varies  from  13  to  18  per  cent. 

Caffeine  -  Tannate.  —  Experiments  in  the  "Lancet" 
laboratory  (Lancet,  1911,  vol.  i,  page  46 ;  and  vol.  ii,  page 
I573)  ted  to  the  conclusion  "  that  an  infusion  of  tea  is  a 
solution  of  caff eine-t annate  in  an  alkaline  medium."  On 
neutralization  (with  HC1)  the  caffeine-tannate  is  gradually 
thrown  out  of  solution,  it  being  only  slightly  soluble  in 
cold  water  but  readily  soluble  in  hot  water.  Caffeine- 
tannate  is  a  compound  of  one  part  of  caffeine  with  three 
parts  of  tannic  acid.  When  these  substances  are  present 
in  these  proportions  in  a  tea  infusion,  they  neutralize  each 
other's  effects  by  combination,  and  are  precipitated  by 
the  gastric  juice.  Excess  of  either  is  detected  by  the 
palate  as  bitterness  or  astringency  in  the  infusion. 

Aroma  is  mainly  due  to  a  volatile  oil,  which  is  present 
to  the  extent  of  0-5  per  cent. 


BEVERAGES  117 

Microscopic  Characters  are  important.  Soak  leaves  in 
hot  water,  when  they  are  easily  laid  out  flat  on  a  slide,  and 
examined*  The  tea  leaf  has  a  serrated  margin  (but  not 
quite  to  the  stalk),  the  primary  veins  run  out  from  the 
midrib  nearly  to  the  margin  and  then  loop:  possesses 
stomata  and  long  hairs  on  the  under  surface,  and  internally 
shows  long  branched  cells,  called  "  idioblasts."  The  apex 
of  the  leaf  is  described  as  notched  or  emarginate.  Average 
size,  i  in.  to  2  in.  by  J  in.  to  1  in. 

Fresh  v.  Exhausted  Leaves. — 

Total  Ash  Soluble  Ash        Alkalinity — KaO 

Fresh  4-8  to  7%     2-8  to  4  %     1-3  to  2% 

Exhausted       4-4  %  07  %  0-2  % 

COCOA. 

Cocoa  is  the  prepared  seed  of  Theobroma  cacao  and  allied 
trees,,  growing  in  the  West  Indies,  Mexico,  Brazil,  etc. 
The  seeds  are  contained  in  a  pod  packed  in  a  pulpy 
substance.  Each  pod  holds  25  to  30  seeds.  The  ripe  pods 
are  allowed  to  ferment,  when  the  seeds  are  easily  separated. 
These  are  then  dried  in  the  sun,  or  in  ovens,  roasted  in  iron 
cylinders,  cracked  by  machinery,  and  the  husks  separated 
from  the  cocoa  beans  or  "  nibs."  The  nibs  are  then 
ground,  but  either  some  of  the  fat  has  to  be  removed  or 
some  diluent  (as  starch  or  sugar)  added  before  they  can 
be  made  into  a  powder.  A  further  treatment  is  to  add 
alkali,  which  may  be  potassium  carbonate  or  the  sodium 
or  ammonium  salt.  The  added  alkali  emulsifies  the  fat 
and  saponifies  any  free  fatty  acid,  so  that  on  addition  of 
hot  water  there  is  less  tendency  for  the  fatty  globules  to 
separate  the  so-called  "  soluble  cocoa."  Cocoa  contains 
the  following  :  moisture,  fat,  starch,  theobromine,  proteins, 
cellulose,  and  mineral  matter  (including  phosphates). 

Moisture. — Dry  2  grm.  in  water-oven.  In  raw  cocoa, 
4-5  per  cent ;    commercial,  8  to  13  per  cent. 

Fat  is  estimated  by  the  Soxhlet  process ;  51  per  cent  in 
raw  cocoa  and  about  30  per  cent  in  prepared.  It  is  also 
known  as  cacao  butter,  or  oil  of  theobroma  of  the  B.P., 
and  is  used  for  suppositories.  It  is  yellowish  in  colour,  and 
contains  stearin,  olein,  and  theobromine.      Melting  point 


118  PUBLIC    HEALTH    CHEMISTRY 

31  °  to  340  C.  (Distinguish  from  coco-nut  oil,  from  Cocos 
nucifera,  a  tropical  palm.     Melting  point  200  to  260  C.) 

Ash. — Ignite  5  grm.  in  a  platinum  dish  at  a  low  red  heat. 
If  red,  examine  for  iron.  Is  about  3  per  cent  in  raw  cocoa 
(rock  cocoa),  and  5  per  cent  in  commercial  varieties. 
Contains  about  1  per  cent  of  phosphoric  anhydride,  but 
this  varies. 

Soluble  Ash  is  not  determined  from  the  ash  but  from  the 
soluble  extract.     It  should  not  fall  below  2  per  cent. 

Soluble  Extract  (cold  water  extract). — Five  grm.  of  the 
sample  are  rubbed  up  in  a  mortar  with  250  c.c.  of  cold 
water  until  a  smooth  mixture  results.  Shake  at  intervals, 
and  allow  to  stand  over  night.  Filter  50  c.c.  (=  1  gram, 
of  sample)  and  evaporate  to  a  constant  weight  (dryness). 
Should  not  exceed  18  per  cent.  Any  material  excess  is 
probably  due  to  added  sugar.  This  residue  is  now  ignited 
at  a  gentle  heat,  cooled,  and  weighed,  and  the  result  called 
the  "  soluble  ash  "  (better,  the  ext.  ash). 

Theobromine  is  allied  to  caffeine,  being  a  dimethylxan- 
thin,  and  like  it  is  not  precipitated  by  potassic  mercuric 
iodide,  or  by  I  in  KI.  Estimate  by  treating  with  petroleum 
ether ;  remove  latter  on  water-bath ;  extract  with  alcohol ; 
evaporate  alcohol ;  add  water,  and  clarify  with  PbAc ; 
remove  lead  with  H2S,  and  extract  with  chloroform. 
Amount  varies  from  1-3  to  17  per  cent.  Theobromine 
contains  31-1  per  cent  of  nitrogen. 

Proteins.— Estimated  approximately  by  Kjeldahl  process. 
Deduct  N  due  to  theobromine  and  X  by  6-25.  Averages 
2-2  per  cent  except  where  proteins  added  (plasmon  cocoa) . 

Starch.1 — Cocoa  contains  about  10  per  cent  of  natural 
starch.  The  granules  are  small  and  round  and  are  easily 
distinguished  from  any  likely  to  be  added  as  an 
adulterant. 

Sugar  is  determined  by  extracting  10  grm.  in  a  filter- 
paper  cylinder  in  a  Soxhlet  to  remove  the  fat. 
Dry  and  extract  with  alcohol,  which  dissolves  the  sugar 
and  other  matters.  Remove  latter  as  before,  boil  for  ten 
minutes  with  2  per  cent  HC1,  neutralize,  and  estimate  the 
invert  sugar  by  Pavy's  process.  The  residue  contains  the 
starch.     Boil  for  six  hours  with  200  c.c.   of  2  per  cent 


BEVERAGES  119 

H2S04.  The  new  residue,  treated  with  2  per  cent  NaOH 
and  then  weak  HC1,  and  filtered,  gives  "  insoluble  fibre. " 
Adulterants. — Potassium  carbonate,  ground  cocoa-shells, 
red  sanders  wood,  iron  oxide,  sugar,  and  starch.  Micro- 
scopic examination  detects  most  of  these. 

Paraguay  Tea,  known  as  "  mate,"  contains  1  per 
cent  of  theine.     Used  in  South  America. 

Guarana  contains  5  per  cent  of  theine  or  caffeine.  Used 
for  migraine.     From  seeds. 

Kola. — From  the  seeds  of  a  tree  growing  wild  on  the 
West  Coast  of  Africa.  It  contains  2-42  per  cent  of  caffeine, 
little  fat  (o-68  per  cent),  starch  and  sugar  36-5  per  cent,  and 
proteins  6-7  per  cent.  Spurious  kola  nuts  containing  no 
caffeine  are  much  sold. 

Coca. — From  the  leaves  of  Erythroxylon  coca ;  contains 
the  alkaloid  cocaine.  The  reputed  sustaining  powers  of 
coca  leaves  are  not  marked  in  the  case  of  Europeans, 
probably  from  their  ordinary  dietary  being  rich  in 
stimulant  extractives  of  the  xanthin  group.  On  the 
natives  the  effects  are  notable. 

LEMON    JUICE     AND     LIME    JUICE. 

Lemon  Juice — Is  the  expressed  juice  of  the  Citrus 
limonum,  and  is  a  slightly  turbid,  yellowish  liquid,  with  a 
sharply  acid  taste.  The  British  Pharmacopoeia  standard 
is  :  specific  gravity,  1030  to  1040  ;  citric  acid,  30  to  40  grains 
per  fluid  ounce  ;  and  ash,  not  more  than  3  per  cent.  The 
Board  of  Trade  standard  for  lemon  and  lime  juices  is  : 
sp.  gr.  (when  de-alcoholized)  1030,  and  30  grains  per 
ounce  of  citric  acid.  As  found  in  the  Merchant  Service, 
or  in  the  Royal  Navy,  these  juices  have  sugar  added  to 
them,  and  have  1  ounce  of  brandy  added  per  10  ounces  of 
juice,  or  are  pasteurized  at  1450  F.,  or  are  boiled.  The 
alcoholic  juice  keeps  better,  and  freezes  at  a  lower  tem- 
perature. The  latter  point  is  of  importance  in  Arctic 
and  Antarctic  expeditions.  Good  juice  keeps  about  three 
years ;  bad  juice  becomes  turbid,  stringy,  and  muci- 
laginous ;  glucose  and  CO  2  being  formed  from  the  decom- 
position of  the  citric  and  malic  acids  present.     One  ounce 


120 


PUBLIC    HEALTH    CHEMISTRY 


of  lemon  juice  per  head  per  day  must  be  issued  when  a 
vessel  has  been  ten  days  at  sea,  except  when  in  harbour 
if  fresh  vegetables  can  be  had. 

Lime  Juice — Is  the  expressed  juice  of  the  Citrus 
limetta.  Like  lemon  juice,  it  contains  free  citric  acid, 
traces  of  other  organic  acids,  citrates,  sugar,  and  albuminous 
and  mucilaginous  bodies.  The  average  composition  of 
these  juices  is  : — 


Lemon  Juice 
Lime  Juice 


Total 
Solids 


Sugar 


8-8o%j2-3o% 
8-64%   070% 


Citric        Mineral      , 
Acid         Matter      Potash    P«Oa  (sol.) 


4'57  % 
5*6o% 


0'35  % 
o*35  % 


0-15  % 

0-12  % 


O'OIO  % 

0-065  % 


According  to  the  above  standards,  the  percentage  of  citric 
acid  should  be  6-6  to  8-8  per  cent.  Few  juices  reach  the 
higher  figure.  The  important  points  in  the  analysis  are  : 
specific  gravity,  total  solids,  free  citric  acid,  combined 
acidity,  ash,  free  mineral  acid,  tartaric  acid,  alcohol, 
sulphites,  and  salicylic  acid. 

Specific  Gravity. — Lime  juice  is  usually  about  1035  to 
1037  (=  32  grains  per  oz.). 

Total  Solids. — Evaporate  10  c.c.  over  water-bath,  and 
dry  in  oven  to  constant  weight.     Vary  from  5  to  9  per  cent. 

Ash. — Ignite  the  residue.  For  lemon  juice,  should  not 
exceed  3  per  cent,  and  be  neutral. 

Free  Citric  Acid. — Titrate  20  c.c.  with  N/i  or  N/2 
NaOH,  using  phenolphthalein  as  indicator.  1  c.c.  N/i 
NaOH  s=s  0-07  grm.  crystallized  citric  acid,  H3C6H.07.H70. 
The  percentage  X  4*375  =  grains  per  fl.  oz.  Varies  from 
5  to  9  per  cent. 

Combined  Acids. — The  neutralized  juice  from  the  above 
process  is  evaporated  to  dryness  on  the  water-bath,  and 
the  residue  ignited  at  a  low  temperature.  The  citrates 
are  changed  to  carbonates.  Cool,  and  extract  mass  with 
hot  water,  add  sufficient  N/i  sulphuric  acid  to  make  acid 
(noting  the  quantity),  boil  to  get  rid  of  C02  and  then 
filter.  Now  titrate  with  N/i  NaOH,  using  methyl-orange 
as  indicator,  to  estimate  amount  of  N  /i  sulphuric,  added 
in  excess.     This  amount  deducted  from  the  total  amount 


BEVERAGES  121 

of  sulphuric  used,  gives  the  amount  of  N/i  sulphuric 
required  to  decompose  the  carbonates  formed  from  the 
citrates  by  ignition.  These  citrates  are  partly  present  as 
such,  and  partly  from  neutralization  of  the  free  acid  by 
soda,  some  of  which  is  not  citric.  The  result,  therefore, 
gives  the  total  organic  acid,  free  and  combined,  in  the 
sample,  calculated  as  citric  acid.  The  combined  acid 
expressed  as  citric  acid,  is  got  by  subtracting  from  the 
total  acid  the  amount  of  free  acid  in  sample.  I  c.c.  N/i 
sulphuric  acid  =*  0-07  grm.  crystallized  citric  acid. 

Free  Mineral  Acid. — Place  several  drops  of  methylene- 
violet  solution  on  a  white  slab.  Add  a  drop  of  juice  ; 
if  free  mineral  acid  is  present,  a  greenish  coloration  is 
got.  With  logwood  solution  dried  on  a  slab,  a  drop  of 
juice,  if  it  contains  free  mineral  acid,  gives  a  red  coloration. 

Sulphites. — Add  Zn  and  HC1,  and  heat ;  if  sulphites 
are  present,  H  2S  is  given  off,  and  will  blacken  lead  acetate 
paper  (moist).  Quantitatively  :  take  50  c.c.  of  sample 
and  add  25  c.c.  N/i  KOH,  and  shake  at  intervals  for 
fifteen  minutes.  Add  10  c.c.  25  per  cent  sulphuric  and 
starch  solution,  and  titrate  rapidly  with  N/10  iodine 
until  a  permanent  blue  colour,  lasting  two  minutes,  is 
produced.  1  c.c.  N/10  iodine  =  0-0032  grm.  S02.  When 
the  sulphurous  acid  is  present  in  the  free  state,  a  known 
excess  of  N/10  iodine  may  be  added  to  a  measured  or 
weighed  quantity  of  the  sample,  the  whole  allowed  to 
stand,  with  occasional  shaking,  for  one  hour,  and  then 
titrated  with  standard  thiosulphate  solution  to  find 
excess  of  iodine.  Another  process  for  sulphites,  is  to  take 
a  quantity  of  the  sample  in  a  flask,  add  a  large  excess  of 
water,  and  then  HC1.  Distil  over  the  liberated  SO  2  into 
a  known  quantity  of  N/10  iodine,  and  find  excess  of 
iodine  solution  used  by  titration  with  standard  thiosulphate. 
The  difference  is  the  amount  of  N  /io  iodine  used  in  oxidiz- 
ing sulphurous  acid  to  sulphuric. 

H2S03  +  I«  +  H20  =  H2S04  +  2HI. 

Alcohol. — By  distillation  (see  under  "Beer,"  page  128). 

Salicylic  Acid. — Precipitate  albuminous  matters  with 
lead  acetate  solution ;  filter,  extract  filtrate  several  times 
with  ether,  evaporate  ether,  and  dissolve  residue  in  distilled 


122  PUBLIC    HEALTH    CHEMISTRY 

water.  Test  with  ferric  chloride  ;  a  violet  coloration,  not 
discharged  *by  acetic  acid,  is  positive. 

Tartaric  Acid. — Neutralize  with  NaOH,  add  calcium 
chloride,  and  shake  well :  if  tartaric  is  present,  a  white 
crystalline  precipitate  of  calcium  tartrate  falls.  To  estimate, 
take  25  c.c,  add  potassium  acetate  and  alcohol,  stand  for 
some  hours,  stirring  occasionally,  filter,  washing  with 
saturated  solution  of  potassium  acid  tartrate,  dry,  and 
weigh  as  cream  of  tartar,  KHC4H406,  or  KHT. 

Poisonous  Metals    Test  for  as  on  page  124. 

VINEGAR. 

Vinegar  may  be  defined  as  the  "  product  of  the  alco- 
holic and  acetous  fermentation  of  a  vegetable  infusion." 
This  definition  includes  all  brewed  vinegars,  but  excludes 
wood  vinegar,  made  by  diluting  acetic  acid  derived  from 
the  destructive  distillation  of  wood.  The  vinegars  in 
common  use  are  :  malt,  cider,  wine,  white  or  distilled,  and 
wood  vinegars,  and  vinegar  from  starch,  glucose,  and 
molasses.  The  essential  constituent  is  acetic  acid,  which 
should  not  be  under  3  per  cent  in  a  good  vinegar.  The 
other  constituents,  and  their  amount,  vary  with  the  mode 
of  production  :  malt  vinegar  yielding  the  most,  and  distilled 
vinegar  the  least,  extract  or  total  solids. 

Malt  Vinegar  is  made  from  malted  barley,  which  is  first 
fermented  to  produce  alcohol  (by  yeast),  and  the  resulting 
liquid  poured  over  piles  of  birch  twigs,  and  exposed  to  the 
air.  The  vinegar  plant  (Mycoderma  aceti)  grows  on  the 
twigs,  and  converts  the  alcohol  to  vinegar,  with  formation 
of  small  quantities  of  acetic  ether,  aldehyde, '  and  other 
bodies,  which  give  such  vinegar  its  pleasant  odour. 
Genuine  malt  vinegar  has  the  following  composition  : 
specific  gravity  at  15-5°  F.  1019  ;  acetic  acid,  5-50  per  cent  ; 
extract,  2-50  per  cent  ;  ash,  0-50  per  cent  ;  N,  0-08  per 
cent ;   P205,  0-08  per  cent.     It  is  dextro-rotatory. 

Wine  Vinegars  are  made  from  grape  juice  and  inferior 
new  wine,  and  vary  in  colour  from  straw  to  red  (malt 
vinegar  is  brown).  They  usually  contain  1  per  cent  of 
alcohol,  1  per  cent  of  extract,  5  to  6  per  cent  of  acetic  acid r 
not  less  than  0-25  per  cent  of  ash,  and  small  quantities  of 


BEVERAGES  123 

tartaric  acid  and  KHT.  The  specific  gravity  varies  from 
1015  to  1022. 

Cider  Vinegar,  from  apple  juice,  is  much  used  in  America. 
It  contains  5  per  cent  of  acetic  acid,  some  malic  acid,  is 
laevo-rotatory,  and  contains  no  aldehyde. 

White  Vinegar  is  made  by  distilling  malt  vinegar. 

Wood  Vinegar,  by  destructive  distillation  of  wood. 

Vinegars  from  starch  and  glucose  run  the  same  danger 
of  arsenical  contamination  as  beer  made  from  these 
substances.     They  are  dextro-rotatory. 

Vinegar  was  formerly  excisable,  and  to  preserve  it, 
sulphuric  acid  was  allowed  to  be  added,  not  exceeding 
i/ioooth  part  by  weight  (duty  abolished  in  1844). 

Sodium  carbonate  or  ammonia  gives  a  purplish  precipitate 
in  wine  vinegar,  but  not  in  malt  vinegar. 

Adulterations  and  Contaminations. — Water,  mineral 
acids,  pyro-ligneous  acid,  metals,  colouring  agents,  preser- 
vatives, gypsum,  capsicum,  vinegar  eels.  Vinegar  is  at 
times  pasteurized  at  1450  F. 

Analysis. — Specific  gravity,  total  solids,  ash,  acetic 
acid,  free  mineral  acid,  poisonous  metals,  nitrogen,  and 
phosphoric  acid.     Microscope  for  eels  {Anguillula  oxyphila). 

Specific  Gravity. — By  sp.-gr.  bottle  or  Westphal  balance, 
1015  to  1022.  The  sp.  gr.  of  an  artificial  vinegar  will  vary 
with  the  amount  of  acetic  acid,  caramel,  and  malt  vinegar 
it  contains.  Thus  the  acidum  aceticum  dilutum  of  the 
B.P.,  which  contains  4-27  per  cent  (by  weight)  of  acetic 
acid,  and  should  yield  no  residue  on  evaporation,  has  a 
sp.  gr.  of  1006.  The  sp.  gr.  of  these  vinegars  will  therefore 
average  above  this  figure. 

Total  Solids. — Evaporate  25  c.c.  to  constant  weight  in 
a  tared  dish.  Varies  with  the  vinegar  :  malt,  2-50  per  cent ; 
cider,  2  per  cent ;  wine,  1  per  cent  ;   white,  0-2  or  less. 

Ash. — Ignite  at  a  low  temperature.  Should  be  alkaline 
if  free  from  mineral  acid.  Varies  from  0-5  to  0*2  per  cent 
in  malt  and  cider,  to  0-03  per  cent  or  less  in  distilled. 

Alkalinity  of  Ash. — Extract  ash  with  hot  water,  titrate 
with  N/10  acid,  using  methyl-orange  as  an  indicator. 
1  c.c.  N/10  acid  =  0-0047  &rm-  KgO.  About  0-026  per 
cent  in  malt,  and  0-14  per  cent  in  cider  vinegar. 


124  PUBLIC    HEALTH    CHEMISTRY 

Acetic  Acid. — Take  10  c.c.  of  sample  in  a  porcelain  dish. 
If  dark  coloured,  dilute  well.  Titrate  with  N/2  NaOH, 
using  phenolphthalein  as  indicator:  i  c.c.  N/i  NaOH  = 
o-o6  grm.  glacial  acetic  acid.  Should  not  be  less  than 
3  per  cent. 

Malic  Acid — in  genuine  cider  vinegar.  Gives  a  fairly 
copious  precipitate  with  lead  subacetate  solution. 

Free  Sulphuric  Acid. — A  few  drops  of  sample  are  taken 
on  a  white  slab.  Place  near  a  drop  of  methyl-violet  and 
bring  into  contact  with  a  glass  rod.  If  only  a  trace  of  free 
acid  is  present,  the  violet  changes  to  blue  ;  but  if  more  than 
i  part  per  iooo  be  present,  a  green  colour  develops.  Or 
test  with  logwood,  as  described  under  "  Lime  Juice."  Or 
add  five  drops  of  methyl-violet  solution  to  5  c.c.  of  sample, 
and  compare  with  control.    Dilute  sample  if  dark  coloured. 

Nitrogen. — Evaporate  25  c.c.  to  dryness,  and  proceed  as 
usual  (none  in  wood  or  distilled  vinegars).  Less  than  o-i 
per  cent  as  a  rule. 

Phosphoric  Acid  is  estimated  by  Stock's  method.  This 
consists  in  treating  the  ash  with  nitric  acid,  adding 
ammonia,  then  more  nitric,  till  the  precipitate  formed 
dissolves;  then  more  ammonia,  until  it  returns  slightly. 
Some  fuming  nitric  is  then  added,  the  solution  warmed  to 
700  C,  and  ammonium  molybdate  solution  added,  when  a 
yellow  precipitate  forms  and  is  collected  and  weighed. 
Weight  X  0-0373  pa  P205.     (None  in  distilled  vinegars.) 

Poisonous  Metals. — Dissolve  ash  in  boiling  distilled 
water,  or  this  failing,  in  boiling  dilute  HC1,  or  if  necessary, 
in  HC1  (2  parts)  +  HN03  (1  part),  and  then  test  by  usual 
analytical  table.  HC1  precipitates  Ag,  Hg',  Pb  ;  H  2  S 
precipitates  Pb,  Hg",  Cu,  Bi,  Cd,  As,  Sb,  Sn,  Au,  Pt ;  AmCl 
and  AmOH  precipitate  Al,  Cr,  Fe  ;  Am  2  S  precipitates  Zn 
Mn,  Ni,  Co  ;  Am  2CO  3  precipitates  Ba,  Sr,  Ca  ;  AmHPO  4 
precipitates  Mg. 

Copper,  if  present  in  the  vinegar  of  pickles,  may  be 
detected  by  inserting  the  bright  blade  of  a  steel  knife. 

Arsenic  may  be  derived  from  arsenical  malt,  caramel, 
or  glucose.  Use  Reinsch's  test  (see  under  "  Beer,"  page  129). 

Copper,  lead,  and  tin  may  all  be  derived  in  the  process  of 
manufacture  (cider  vinegar)  or  from  storage  in  metallic 
or  metallic-covered  vessels. 


BEVERAGES  125 

Potassium  ferrocyanide  is  sometimes  used  to  clarify  vine- 
gars,  and  is  said  to  be  decomposed,  leaving  in  solution  an 
unstable  compound  of  prussic  acid  with  the  organic  matter. 

Estimation  of  the  Free  Sulphuric  Acid. — Take  50  ex.  of 
sample,  add  25  c.c.  N/10  NaOH.  Evaporate  whole  to 
dryness,  and  ignite.  To  ash  add  25  c.c.  N/10,  HC1  or 
H2S04,  25  c.c.  aq.  dest.,  and  boil  to  expel  C02.  Filter, 
wash  paper  with  hot  water,  and  titrate  whole  nitrate  with 
N/10,  NaOH,  and  phenolphthalein.  The  number  of  c.c. 
of  soda  used  x  0-0049  —  ^ree  sulphuric  acid  in  50  c.c.  of 
sample. 

Barium  chloride  cannot  be  used  to  test  for  the  free  acid, 
as  vinegar  is  often  made  from  hard  waters  containing 
sulphates,  which  would  also  precipitate  the  barium. 

Microscope  for  vinegar  eels.     (1  to  2*5  mm.  in   length.) 

Preservatives. — Test  as  elsewhere. 

Vinegar,  in  doses  of  from  a  half  to  one  ounce  daily,  is  an 
anti-scorbutic,  but  is  inferior  to  lime  and  lemon-juices. 

BEER. 

Beer  is  usually  defined  as  "  a  fermented  infusion  of 
malt,  flavoured  with  hops,"  but  this  definition  must  be 
extended  to  include  beers  brewed  from  saccharine  fluids 
other  than  malt  infusion,  and  flavoured  with  other  bitters 
than  hops.  The  malt  substitutes  are  such  as  malted 
Indian  corn,  and  starches  chemically  converted  into 
sugars  (rice,  potato,  and  other  starches)  by  the  use  of 
sulphuric  acid.  The  latter  may  contain  arsenic  derived 
from  the  acid  used,  and  so  contaminate  the  beer.  This 
happened  in  South  Lancashire  in  1900.  The  hop 
substitutes  are  used  only  in  a  few  breweries,  of  which  a 
list  is  published  by  the  Inland  Revenue  authorities.  They 
are  such  bitters  as  quassia,  gentian,  calumba,  chiretta. 
Noxious  bitters  have  at  times  been  used,  as  nux  vomica, 
picrotoxin,  and  picric  acid. 

Malt  Beer  is  thus  made : — Barley  grain  is  shot  into  a 
cistern  and  covered  with  water  to  the  depth  of  5  inches 
and  soaked  for  40  to  60  hours,  until  it  has  swollen  up 
and  the  process  of  germination  has  started.  The  wet  grain 
is   then   removed   from   the   steeping-tanks,   piled  up  in 


126  PUBLIC    HEALTH    CHEMISTRY 

heaps,  called  "  couches,"  and  allowed  so  to  remain  for 
20  to  48  hours,  to  enable  it  to  develop  sufficient  heat  to 
ensure  equable  and  good  germination.  Thereafter  it  is 
spread  out  on  floors,  where  rapid  germination  proceeds, 
the  plumule  growing  up  the  back  of  the  grain  and  the  tiny 
rootlets  being  put  forth.  In  10  to  14  days  the  plumule  is 
halfway  to  three-quarters  up  the  back  of  the  grain.  Further 
growth  is  then  stopped  by  running  the  malt  on  to  the 
floors  of  the  "  kilns,"  where  it  is  gradually  heated  until 
dry,  when  the  heat  is  increased.  If  pale  malt  is  desired, 
the  temperature  is  not  raised  above  1850  F.,  but  for  black 
or  brown  malts  for  black  beers,  the  malt  is  caramelized. 
The  heated  malt  is  then  sifted  or  screened,  to  get  rid  of  the 
sproutlings  ;  the  sif tings  are  ground  into  "  grist,"  and  the 
grist  is  run  into  the  "  mash-tun,"  a  large  covered  vessel, 
containing  a  proper  proportion  of  the  brewing-water,  at  a 
temperature  of  1400  to  1520  F.  for  pale  ales,  and  1440  to 
1500  F.  for  black  beers.  In  the  mash-tun  it  is  well  mixed  for 
15  to  30  minutes,  allowed  to  stand  for  two  hours  more,  and 
then  the  liquor,  or  "  wort,"  run  off  into  a  receiving  vessel. 
More  water  is  run  into  the  mash-tun,  and  the  solids  are 
washed  or  "  sparged,"  the  water  not  being  used  hotter  than 
1600  F.  to  prevent  the  solution  of  starch.  After  four  hours' 
sparging  the  liquor  is  run  off,  the  speed  being  increased 
as  the  solids  settle,  and  is  mixed  with  the  first  wort.  The 
mixture  is  then  boiled  in  the  "  copper,"  the  larger  portion 
of  the  hops  added,  and  after  one  to  two  hours'  boiling,  run 
out  and  the  hops  allowed  to  settle.  The  bright,  clear, 
supernatant  liquid  is  then  run  into  large  flat  trays  or 
coolers,  and  over  coils  of  pipes  in  which  cold  water  is 
circulating-  (refrigerators),  its  temperature  being  reduced 
to  a  suitable  one  for  fermentation  (6o°  F.  or  under).  At 
this  stage  the  specific  gravity  of  the  wort  is  taken  by  the 
Excise  officers  with  a  Bates'  saccharometer,  which  is  a 
modified  hydrometer.  The  Customs'  standard  is  a  sp.  gr. 
of  1055  at  60  °  F.  After  allowing  6  per  cent  off  for  wastage, 
they  charge  7s.  o,d.  per  36  gallons,  as  duty.  The  wort  is 
now  run  into  tuns  made  of  wood,  slate,  or  stone,  about 
1  lb.  of  yeast  per  barrel  being  mixed  with  it  as  it  is  run  in. 
Fermentation  now  takes  place,  and  is  continued  until  the 
beer  reaches  the  specific  gravity  desired  by  the  brewer. 


BEVERAGES  127 

The  yeast  is  removed  by  skimming  or  otherwise,  and  the 
beer  run  into  barrels.  When  about  to  be  sent  out  to  the 
"  trade,"  it  is  subjected  to  "  fining."  Finings  are  made 
of  isinglass,  and  are  white  or  brown.  They  are  added  in 
the  proportion  of  i  to  2  pints  per  barrel ;  4  lb.  of  isinglass 
dissolved  in  a  hogshead  of  weak  acid  or  sour  beer  con- 
stitutes the  usual  finings  (hogshead  =  63  wine,  or  52-5 
imperial  gallons). 

Beers  from  starches,  glucose,  invert  sugar,  etc.,  are 
similarly  treated,  beginning  with  the  mash-tun. 

Brewing- Waters. —  A  hard  water  free  from  nitrates 
and  organic  matter  is  preferred  for  brewing.  It  should 
also  contain  10  to  15  grains  per  gallon  of  NaCl.  The  com- 
position of  Burton  water  is  taken  as  a  standard  in  this 
country,  and  it  contains  11  to  15  grains  of  carbonates  of 
lime  and  magnesia,  40  to  60  of  sulphate  of  lime,  12  to  30  of 
sulphate  of  magnesia,  and  5  of  chloride  of  Na  and  K.  In 
the  Dublin  water  the  carbonates  are  about  11  grains  per 
gallon,  the  sulphates  only  o-8,  and  the  chlorides  i-8.  The 
sulphates  and  carbonates  of  Na  and  K  are  injurious,  as 
are  also  any  iron  salts.  The  hard  water  does  not  take 
up  so  much  albuminous  matter  from  the  malt.  Nitrates 
restrain  the  growth  of 'the  yeast.  Organic  matter  in  the 
water  spoils  the  keeping  quality  of  the  beer. 

Yeast. — In  England  the  "  high  "  or  surface  fermentation 
is  used  (top  yeast)  ;  in  Germany  the  "  low"  or  sedimen- 
tary process  (bottom  yeast) .  The  latter  works  at  a  lower 
temperature,  and  gives  a  more  gaseous  but  less  alcoholic 
beer. 

Composition. — Beer  consists  of  water,  alcohol,  maltose, 
dextrin,  albuminoids,  bitters,  salts,  carbonic  acid  gas, 
and  acidity  (acetic,  lactic,  succinic,  etc.).  Preservatives 
(salicylic  acid,  boric  acid),  saccharin  (prohibited),  and 
arsenic  may  be  present.  All  the  constituents  vary  greatly 
in  the  different  makes. 

Analysis. — The  beer  is  shaken,  and  poured  from  one 
vessel  to  another  to  get  rid  of  the  CO  2  gas,  and  the  froth 
is  got  rid  of  by  filtering  through  cotton-wool.  Specific 
gravity :  by  bottle  or  Westphal,  varies  from  1006  to  1030 
(Bass  1013).      Extract :    evaporate  5  c.c.  to  dryness.      See 


128  PUBLIC    HEALTH    CHEMISTRY 

also  under  Addenda,  page  130.  (Bass  7  per  cent).  Ash  : 
ignite  extract  at  a  low  temperature.  Under  0-5  per  cent. 
Sodium  chloride :  extract  ash  with  water  and  titrate  with 
standard  AgN03. 

Acidity. — Total.  Titrate  20  c.c.  of  sample  (well  diluted) 
with  N/10  NaOH  and  litmus,  and  note  number  of  c.c. 
required.  Calculate  as  glacial  acetic  acid  (1  c.c.  N/10 
soda  =  0-006  grm.  Ac).  Is  commonly  0-182  per  cent,  or  16 
grains  per  pint. 

Fixed  : — Dilute  20  c.c.  to  100  c.c.  and  evaporate  down  to 
50  c.c.  Dilute  again,  and  titrate  with  N/10  soda.  1  c.c.  — 
0-009  §rm-  lactic  acid.     Calculate  to  a  percentage. 

Volatile  : — Deduct  number  of  c.c.  required  for  fixed  acid 
from  number  required  for  total,  and  calculate  as  acetic. 

Alcohol. — Two  methods  : — 

1.  By  direct  distillation. — 100  c.c.  of  sample  are  taken  in 
a  distilling-nask,  a  small  piece  of  pumice  stone  is  added, 
and  about  90  c.c.  are  distilled  over.  The  distillate  is  cooled 
to  15-5°  C,  and  made  up  with  aq.  dest.  to  100  c.c.  at  the 
same  temperature.  The  specific  gravity  is  now  taken  by 
the  bottle  or  Westphal  balance.  The  amount  of  alcohol 
is  then  found  from  tables. 

2.  Indirect  or  Tabarie's  method. — Take  sp.  gr.  of  sample 
carefully  (at  15-5°  C).  Take  100  c.c.  and  evaporate  down 
to  one-third  of  the  bulk  to  get  rid  of  alcohol  and  other 
volatile  substances.  Cool,  make  up  to  original  bulk  at 
I5"5°  C.,  and  take  sp.  gr.  again.  Then  the  original  sp.  gr. 
-*■  new  sp.  gr.  =  sp.  gr.  of  an  alcoholic  water  of  same  strength 
as  beer.  Find  percentage  from  tables.  Or.  deduct 
difference  between  the  two  specific  gravities  from  1000,  and 
the  result  is  sp.  gr.  of  an  alcoholic  water  of  same  alcoholic 
strength  as  the  sample  of  beer  (average  5  per  cent). 

Original  Gravity  of  Beer  Wort. — This  is  sometimes 
needed  to  calculate  the  rebate  or  drawback  of  duty  allowed 
when  beer  is  exported.  It  is  obtained  by  deducting  the 
sp.  gr.  of  the  alcoholic  distillate  in  (1)  above  from  1000. 
The  number  got  is  the  "  spirit  indication,"  and  from  a 
table  we  get  with  its  help  the  number  of  degrees  of  "  gravity 
lost."  This  number,  added  to  the  sp.  gr.  obtained  by 
Tabarie's  method  for  the  de-alcoholized  beer,  gives  the 
original  gravity  of  the  beer  wort.     A  further  addition  for 


BEVERAGES  129 

excess  of  acidity  (over  o-i  per  cent  acetic)  is  also  made 
by  consulting  another  table.     (Bass,  1056). 

Saccharin. — Treat  dried  extract  with  anhydrous  ether ; 
evaporate  to  dryness  ;  if  residue  sweet,  infer  presence  of 
saccharin. 

Preservatives. — Boric  acid:  test  ash  (as  on  page  80). 
Salicylic  acid:  kiln-dried  malt  contains  a  principle  which 
gives  an  identical  reaction  with  ferric  chloride.  Hence 
use  Spica's  test  :  Take  100  c.c.  of  beer,  acidify  with 
H  2SO  4  ;  extract  with  ether  ;  separate  ether  ;  evaporate 
spontaneously ;  and  warm  residue  carefully  with  a  drop 
of  strong  HNO3.  If  salicylic  acid  is  present,  picric  acid 
is  formed.  Add  AmOH  or  NaOH,  and  a  bright-yellow 
picrate  is  formed,  which  will  stain  a  woollen  thread 
immersed  in  it. 

Arsenic. — By  Reinsch's  test.  Take  200  c.c.  of  sample, 
add  30  c.c.  strong  HC1  and  a  piece  of  clean  bright  copper 
foil  I  in.  x  J  in.  Boil  for  forty-five  minutes,  adding  aq. 
dest.  to  keep  up  bulk  as  required.  Cool,  and  examine 
copper  foil.  If  clean  and  bright  copper-coloured,  As,  Sb, 
and  Hg  are  absent.  If  changed,  there  may  be  A s  or  Sb 
if  deposit  is  black  ;  but  if  silvery,  it  is  Hg.  Wash  gently 
with  water,  alcohol,  and  ether  in  succession.  Dry  at  ioo°  C. 
Put  in  a  perfectly  dry  2-in.  glass  reduction  tube,  the  upper 
part  of  which  has  been  previously  warmed.  Heat  gently. 
As,  Sb,  and  Hg  all  sublime  and  condense  in  the  upper  part 
of  the  tube  as,  respectively,  As  20  3  (crystals) ;  Sb  20  3 
(amorphous)  ;  and  Hg  (globules).  Examine  sublimate 
magnified  200  times.  Arsenic  gives  octahedral  crystals. 
Antimony  gives  an  amorphous  mass.  Mercury  gives 
globules.  If  arsenic  found,  wash  out  sublimate  with 
weak  KOH  solution  and  put  two  portions  on  a  white  slab  : 
(1)  Touch  one  with  ammonia-cupric  solution,  when 
Scheele's  green  is  formed  ;  (2)  Touch  other  with  ammonia- 
silver-nitrate  solution  —  canary-yellow  precipitate.  If 
antimony  found,  wash  out  with  weak  tartaric  acid,  which 
forms  tartrated  antimony.  Add  HC1  and  H2S — orange 
precipitate.  If  mercury  found,  expose  to  iodine  vapour — 
yellow  iodide  formed.  A  control  should  be  done  with  the 
distilled  water  and  the  HC1  and  copper  foil. 

Marsh's  and  Gutzeit's  tests  are  also  used. 

9 


130  PUBLIC    HEALTH    CHEMISTRY 

Addenda. — A  pint  of  good  beer  contains  roughly  i  fl.  oz. 
of  alcohol,  1-5  oz.  of  sugary  extract,  20  grains  of  free  acid, 
14  grains  of  salts,  and  1  pint  of  CO  2  gas  in  solution.  The 
extract  may  be  got  from  the  sp.  gr.  of  the  de-alcoholized 
beer  by  dividing  the  excess  over  1000  by  3-86.  The 
difference  between  the  sp.  gr.  of  the  beer  and  the  sp.  gr. 
of  the  de-alcoholized  beer  may  be  taken  as  the  approximate 
spirit  indication  in  calculating  the  original  gravity  of  the 
beer  wort,  and  deducted  from  1000,  gives  sp.  gr.  of  alcoholic 
water  of  same  alcoholic  strength  as  beer. 

WINE. 

Wine  is  the  fermented  juice  of  the  grape,  but  much 
of  it  is  made  otherwise.  The  grapes  are  pressed  or 
trodden  (mechanical  pressure  extracts  an  excess  of  tannin 
and  colouring  matter  from  red  grapes),  and  the  juice  or 
"  must  "  ferments  spontaneously  from  the  yeast  existing 
naturally  on  the  skins,  the  glucose  or  grape  sugar  being 
converted  into  alcohol  and  C02.  When  fermentation  has 
ceased,  the  wine  is  run  off  from  the  residue  or  "  lees," 
composed  mainly  of  yeast  cells  and  cream  of  tartar  (KHT). 
The  wine  is  kept  in  casks  in  which  a  further  fermentation 
takes  place,  resulting  in  the  deposition  of  more  KHT.  It  is 
then  put  into  casks  to  mature  or  "  age."  In  making  white 
wine  the  must  is  separated  from  the  skins  and  stalks,  while 
for  red  wine  the  skins  of  purple  grapes  are  fermented  with 
the  must.  In  grapes  the  relative  amount  of  sugar  and 
albuminous  matter  varies.  The  yeast  fungus  lives  on  the 
latter,  and  if  it  is  used  up  before  all  the  sugar  is  fermented 
a  sweet  wine  results  ;  if  the  contrary  happen,  a  non-sweet 
or  "  dry  "  wine  is  obtained.  The  grape  juice  also  contains 
tartaric  acid  and  its  salts,  and  the  proportion  of  acid  to 
sugar  best  adapted  for  wine  production  is  1  to  40.  In  some 
parts,  if  the  acid  is  in  excess,  the  must  is  diluted,  and  sugar 
(glucose  or  cane  sugar)  added  in  the  necessary  amount. 
To  make  dry  wines,  white  of  egg,  gelatin,  or  isinglass  is 
added  to  feed  the  fungus  until  it  ferments  all  the  sugar. 

Analysis. — 

Specific  gravity :  varies.     Extract. — 2-4  per  cent. 

Alcohol. — By  distillation  :  6  to  17  per  cent. 


BEVERAGES  131 

Ash. — o-i  to  0-3  per  cent.     About  one-sixth  is  P205. 

Acidity. — (i)  Fixed  as  tartaric.  Dilute  20  c.c,  boil  down, 
repeat,  add  water,  and  titrate  with  N/10  NaOH,  using 
phenolphthalein  as  indicator  (0-5  per  cent  and  under)  ; 
(2)  Volatile  acid  :  20  c.c.  of  the  wine  are  well  diluted,  and 
then  titrated  with  N/10  soda,  using  phenolphthalein  as 
before.  Deduct  number  of  c.c.  required  for  fixed  acid 
from  number  now  obtained,  and  multiply  difference  by 
0-006  ==  volatile  acid  as  acetic.  The  fixed  acidity  is 
calculated  as  tartaric  :  1  c.c.  N/10  NaOH  =  0-0075  grm. 
tartaric  acid. 

Sugar. — Take  50  to  100  c.c,  boil  off  the  alcohol,  remove 
colouring  matter  and  other  bodies  with  slight  excess  of 
basic  lead  acetate,  filter,  remove  lead,  and  treat  with 
Fehling's  solution  after  dilution  to  200  c.c.  Varies  from 
o  to  5  per  cent ;   in  champagne,  4  to  10  per  cent. 

Preservatives. —  Salicylic  acid,  formaldehyde,  sulphites. 
Boric  acid  is  said  to  be  naturally  present  in  some  wines. 

Colouring  Matter. — Soak  gelatin  (10  per  cent)  cubes  in 
sample  for  twenty-four  hours,  then  cut  them  diagonally. 
Natural  wine  colour  penetrates  less  than  one-eighth  of  an 
inch,  but  artificial  colours  through  and  through.  Or,  the 
Paris  Municipal  Laboratory  test :  Take  a  piece  of  recently 
calcined  lime  and  wet  it  with  a  few  drops  of  the  wine. 
Natural  red  wine  gives  a  yellowish-brown  coloration  ;  wine 
coloured  with  fuchsin  or  Brazil  wood  gives  a  rose  colour, 
and  wine  coloured  with  logwood  gives  a  reddish  violet.  Or, 
baryta  water  is  added  until  solution  is  green,  and  then  acetic 
ether,  and  shake.  Allow  to  stand  until  acetic  ether  separ- 
ates, when  if  coloured,  basic  dyes  present,  if  not,  probably 
only  natural  colour.  Or,  add  diluted  KOH  until  alkaline, 
then  mercuric  acetate,  filter;  filtrate  is  red  or  yellow  if 
acid  aniline  dyes  present,  colourless  if  pure  wine  colour. 

Adulteration. — Addition  of  tannin,  alum,  catechu; 
plastering  (addition  of  gypsum)  ;  blending  ;  pasteuriza- 
tion ;    saccharin. 

Piquette. — An  artificial  substitute  for  wine,  manufac- 
tured in  France  (50  million  gallons  were  made  and  con- 
sumed in  1898).  One  pound  of  raisins  and  one  pound  of 
dried  apples  are  added  to  one  gallon  of  water  ;  expose 
mixture  in  an  open  vessel  to  the  air  for  three  days ;   then 


132  PUBLIC    HEALTH    CHEMISTRY 

bottle,  adding  one-half  teaspoonful  of  sugar  and  a  small 
piece  of  cinnamon  to  each  bottle. 

Cider. — Is  the  fermented  juice  of  the  apple.  Contains 
about  3  to  5  per  cent  of  alcohol. 

Perry. — The  fermented  juice  of  the  pear  ;  alcoholic 
strength  tends  to  be  higher  than  in  cider. 

SPIRITS. 

Spirits  are  all  made  by  the  distillation  of  alcohol  produced 
by  the  fermentation  of  various  saccharine  or  starchy 
materials,  such  as  fruit  juices,  grain,  and  molasses.  The 
essential  constituent  is  ethyl  alcohol,  but  they  also  contain 
varying  proportions  of  other  alcohols,  ethers,  and  other 
fragrant  bodies,  and  in  some  cases  added  substances. 

Ethyl  Alcohol  (C2H5OH)  is  commonly  denoted  simply 
alcohol.  It  is  a  liquid  which  boils  at  j8°  C,  and  at  200  C. 
has  a  sp.  gr.  of  789.    It  oxidizes  to  acetic  acid  (CH,-COOH.) 

Brandy  is  made  by  the  distillation  of  fermented  grape 
juice.  It  is  at  first  colourless,  but  is  stored  in  casks  which 
give  it  an  amber  colour.  Sp.  gr.  is  usually  about  930,  alcohol 
is  48  to  56  per  cent,  and  total  solids  are  about  o-i  per  cent. 
Besides  alcohol  and  water,  it  contains  traces  of  various 
ethers,  aldehydes,  and  acids  (chiefly  acetic).  Imitation 
brandy  is  made  from  grain  spirit  flavoured  with  various 
esters  and  oils  (cloves,  cassia),  and  coloured  with  caramel. 

Whiskey  or  Whisky  (Gaelic,  uisgebeatha — water  of 
life)  is  made  by  distillation  from  malted  barley,  oats,  or 
rye  which  has  been  fermented.  It  is  stored  in  sherry 
casks  which  flavour  and  colour  it.  Numerous  imitations 
exist,  especially  from  potato  spirit.  Alcohol,  44  to  50  per 
cent ;  total  solids,  0-15  per  cent ;  acidity  as  acetic,  o-i 
per  cent.     Specific  gravity,  935  to  945. 

Rum  is  made  by  the  distillation  of  the  fermented  juice 
of  the  sugar  cane  or  from  molasses  (the  drained  syrup  from 
which  sugar  does  not  crystallize  on  boiling)  and  coloured 
with  caramel.  Its  characteristic  odour  is  due  to  ethyl 
butyrate.  Alcohol,  40  to  50  per  cent ;  total  solids,  07  to 
1*5  per  cent. 

Gin  is  made  by  distillation  from  fermented  grain 
flavoured  with  juniper  berries,   oil  of  turpentine,   oil  of 


BEVERAGES  133 

juniper,  coriander  seeds,  capsicum,  etc.,  with  or  without 
the  subsequent  addition  of  cane  sugar  (to  sweeten  it).  It 
acts  on  the  kidneys.  Alcohol,  30  to  40  per  cent.  Hollands 
and  Schnapps  are  varieties  from  rye. 

Proof  Spirit  is  a  term  in  use  for  excise  purposes,  denoting 
a  dilute  spirit  of  definite  strength.  It  contains  of  absolute 
alcohol  if  expressed  as  (1)  Volume  in  volume,  57-05  per  cent ; 
(1  in  1753)  ;  (2)  Weight  in  volume,  42-46  per  cent  (1  in 
2'355)  i  (3)  Weight  in  weight,  49-25  per  cent  (1  in  2-03). 
Spirits  weaker  than  proof  spirit  are  said  to  be  under  proof, 
and  when  stronger  to  be  over  proof.  Sp.  gr.,  919-8  at  150  C. 
Whisky  containing  60  per  cent  of  alcohol  volume  in 
volume,  is  equal  per  100  measures  to  60  X 1-753  =  105-18 
measures  of  proof  spirit,  and  is  said  to  be  5-18°  over 
proof  ;  but  if  it  contains  only  30  per  cent  of  alcohol  volume 
in  volume,  then  100  volumes  less  52-59  =  4741  under  proof. 

Analysis. — 

Alcohol. — By  direct  distillation  after  diluting  sample 
with  an  equal  bulk  of  water  to  prevent  loss  of  alcohol 
from  too  rapid  evolution  and  imperfect  condensation.  By 
the  Sale  of  Food  and  Drugs  Amendment  Act,  1879, 
brandy,  whisky,  and  rum  must  not  be  weaker  than  250 
under  proof  (=  75  per  cent  proof  spirit),  and  gin  not  weaker 
than  350  under  proof  (=  65  per  cent  proof  spirit). 

Acidity. — The  fixed  may  be  determined  on  the  residue 
from  the  distillation  by  titration  with  N/10  baryta  or  soda, 
and  phenolphthalein.  The  volatile  is  got  by  titrating  the 
sample,  deducting  the  number  of  c.c.  required  for  fixed 
acid,  and  calculating  as  acetic  acid.  The  fixed  is  returned 
as  tartaric  acid.     Spirits  should  not  contain  any  fixed  acid. 

Total  Solids. — Evaporate  on  the  water-bath  and  dry  to 
constant  weight  in  water-oven. 

Ash. — Ignite  residue. 

Ethers  (compound). — These  are  calculated  as  so  many 
parts  per  100,000  of  alcohol,  and  the  usual  amount  in 
brandy  is  about  100,  in  whisky  from  10  to  90,  and  in  rum 
the  extremes  are  as  great  as  30  to  400. 

Furfurol  is  an  aldehyde,  and  is  present  in  pot-still, 
but  not  in  patent-still  spirit.  It  is  tested  for  by  adding 
10  drops  of  colourless  aniline  oil  to  10  c.c.  of  distillate 
(made  down  to  50  per  cent  of  alcohol),  and  1  c.c.  of  acetic 


134  PUBLIC    HEALTH    CHEMISTRY 

acid  (free  from  alcohol).  A  rose  tint  appears,  which  is 
compared  with  that  from  10  c.c.  of  standard  solution  of 
furfurol  (0-005  grm-  Per  litre)  similarly  treated.  From  1 
to  3  parts  per  100,000.     Formula:  C4H3OCHO. 

Fusel  Oil  (Amyl  Alcohols,  C6H„*OH). — Spirits  should 
never  contain  more  than  o-i  per  cent  amyl  alcohol,  as  such. 
Tests : — Distil  off  ethyl  alcohol  from  100  c.c.  on  water- 
bath  and  take  residue.  Add  an  equal  bulk  of  water,  cool, 
and  extract  with  ether.  Let  ether  evaporate  at  room 
temperature  and  divide  residue  into  three  parts. 

1.  Heat  one  portion  with  sulphuric  acid  and  a  little  potas- 
Lium  bichromate  ;   a  smell  of  valerianic  acid  is  obtained. 

2.  Heat  with  sulphuric  acid  and  sodium  acetate,  when 
the  odour  of  jargonelle  pears  (acetate  of  amyl)  is  got. 

3.  Warm  with  double  its  volume  of  strong  sulphuric  acid  ; 
violet-red  colour  of  amyl  sulphuric  acid  is  formed. 

Aldehydes. — Vary  from  10  to  40  parts  per  100  litres  of 
alcohol  (100,000  parts). 

Higher  Alcohols. — From  100  to  250  parts  per  100,000 
parts  of  absolute  alcohol. 

Total  Secondary  Products,  or  "  the  co-efficient  of  im- 
purities," is  the  sum  total  of  the  free  acid,  aldehyde,  fur- 
furol, ethers,  and  higher  alcohols.  According  to  the  Lancet 
Commission  this  co-efficient  varies  for  a  specially  fine 
brandy  from  300  to  646,  bat  may  fall  to  250  per  100,000  of 
absolute  alcohol  in  inferior  but  genuine  brandy.  Grain  and 
beet  spirits  are  comparatively  free  of  secondary  products, 
furfurol  especially  being  absent.  Gin  is  also  low  in  total 
secondary  products.  Jamaica  rum  is  very  high  in  ethers 
(400  grm.  of  ethyl  acetate  per  100  litres  of  absolute  alcohol 
present)  and  contains  more  acids  and  furfurol  than  brandy. 
Whisky  closely  resembles  brandy,  but  the  furfurol  is  high. 

Specific  gravity  is  frequently  ascertained  by  Sykes'  hydro- 
meter, the  temperature  of  the  liquid  being  noted,  and  the 
result  obtained  from  special  tables,  which  give  the  amount 
that  the  sample  is  over  or  under  proof.  If  there  is  much 
solids  the  spirit  must  be  distilled  as  for  beer  and  wine. 

Methyl  Alcohol,  CH3OH  (wood  spirit). — Is  a  liquid 
which  boils  at  66°  C,  and  at  200  C.  has  a  sp.  gr.  of  796. 
From  it  are  derived  formaldehyde  (HCHO)  and  formic 
acid  (H-COOH). 


CHAPTER     VII. 

DISINFECTANTS,    ANTISEPTICS,    AND 
DEODORANTS. 

A  disinfectant  is  an  agent  which  destroys  the  causes  of 
disease  and  their  products,  such  as  fire,  steam  (saturated), 
boiling  water,  hot  air,  and  chemicals  in  a  proper  strength 
(5  per  cent  carbolic  acid,  5  per  cent  permanganate  of 
potash,  o-i  per  cent  of  perchloride  of  mercury,  formalin, 
cyllin,  lysol,  etc.,  and  CI,  Br,  and  I  and  ozone). 

An  antiseptic  is  an  agent  which  arrests  or  impedes  the 
growth  of  micro-organisms  without  destroying  their 
vitality.  Most  of  the  disinfectants  act  thus  in  the  weaker 
strengths,  as  do  also  borax  and  boracic  acid,  sulphites  and 
sulphurous  acid,  salicylates  and  salicylic  acid,  essential 
oils,  quinine  and  other  alkaloids,  and  common  salt. 

A  deodorant  is  an  agent  which  masks  or  destroys  the 
effluvia  produced  by  certain  micro-organisms.  Deodorants 
include  nitrous  acid  fumes,  chlorine  fumes  (from  chloride 
of  lime),  sulphurous  acid  fumes,  fumes  of  wood,  tar,  and 
burnt  paper,  and  some  of  the  disinfectants  in  virtue  of 
their  oxidizing  power  or  their  strong  odour,  such  as 
permanganate  of  potash  and  carbolic  acid. 

Direct  sunlight  and  fresh  air  are  powerful  factors  in,  and 
aids  to,  disinfection,  antisepsis,  and  deodorization. 

Bleaching  Powder  is  prepared  by  passing  CI  gas  over 
slaked  lime,  when  a  compound  is  formed  having  the 
formula  Ca  (OC1)  CI,  which  liberates  CI  gas  on  treatment 
with  acids.  Theoretically  it  should  contain  56  per  cent  of 
available  CI ;  a  good  bleaching  powder  commercially  is 
one  which  gives  35  per  cent  of  available  CI. 

Ca(OCl)Cl  +  2  HC1  =  CaCl  2  +  H  20  +  CI  2. 

Process.     Pinot's  Method. — 

Solutions  required :  (1)  Standard  arsenious  solution. 
Dissolve  4-95    grm.   of    pure    arsenious  oxide    in   about 


136  PUBLIC    HEALTH    CHEMISTRY 

250  c.c.  of  water,  along  with  25  grm.  of  sodic  carbonate. 
Boil  for  some  time  until  all  the  oxide  is  dissolved,  cool,  and 
make  up  to  1  litre.  This  is  decinormal  strength  and  can 
be  standardized  against  N/10  I. ;  (2)  N/10  iodine  solution ; 
(3)  Starch  and  KI  paper  as  indicator.  Take  2-5  grm. 
of  sample  and  rub  up  with  successive  quantities  of 
water  until  all  the  powder  is  transferred  to  a  250  c.c. 
flask  to  which  the  washings  are  added.  Make  up  to  the 
mark,  shake  well,  and  taking  20  c.c.  of  the  milky  fluid, 
titrate  with  the  standard  arsenious  solution  until  starch  and 
KI  paper  is  no  longer  blued  on  being  touched  with  a  drop 
of  the  mixture  removed  on  a  glass  rod.  As  excess  of  the 
arsenious  solution  is  easily  added,  it  is  usual  to  now  add 
starch  to  the  titrated  liquid  and  titrate  with  N/10  iodine 
until  a  permanent  blue  is  got.  The  amount  of  the  N/10  I 
used  is  deducted  xrom  the  amount  of  arsenious  solution 
required,  and  as  1  c.c.  As  20  3  solution  =  0-00355  grm- 
CI,  or  3 -55  mgr.  CI,  the  amount  of  CI  is  easily  calculated 
and  is  returned  as  a  percentage. 

2  Ca(OCl)Cl+ 2  H  20+ As  20  3= As  20  5+2  CaCl  2+ 2  H  20. 

Formalin  is  a  40  per  cent  solution  in  water  of  formalde- 
hyde (CH20),  the  qualitative  tests  for  which  have  been 
given  on  page  80.  Quantitatively  it  is  tested  as  follows  : 
Weigh  out  2-075  grm.  of  formaldehyde  sample,  and  dilute 
with  water  to  500  c.c.  Take  10  c.c.  of  this  dilution  and 
mix  with  25  c.c.  of  N/10  iodine  solution  in  a  flask.  Add 
NaOH  solution  (15  per  cent)  drop  by  drop  until  the  liquid 
becomes  clear  yellow,  and  allow  to  stand  for  ten  minutes. 
Then  add  sufficient  dilute  HC1  to  liberate  the  iodine  not 
acted  on  by  the  formalin,  and  titrate  the  amount  of  I  with 
N/10  thiosulphate ;  the  number  of  c.c.  of  thiosulphate 
required  subtracted  from  25,  gives  the  number  of  c.c.  of 
N/10  I  absorbed.  1  c.c.  N/10  I  =  0-0015  grm.  CH20. 
HCHO  +  H  2  O  +  1 2  =  H-COOH  -f  2HI. 

Permanganate  of  Potash. — The  solution  is  titrated 
with  standard  ferrous  sulphate  solution  in  presence  of 
sulphuric  acid.  Take  5  c.c.or  10  c.c.  of  sample  (the  smaller 
the  amount  the  stronger  the  sample),  add  10  c.c.  of  25 
per  cent  H2S04,  and  from  a  burette  add  standard  FeS04 
solution  until  colourless.     Calculate  to  a  percentage. 


DISINFECTANTS,    ETC.  137 

Ferrous  Sulphate. — Reverse  the  above  process,  using 
a  standard  solution  of  permanganate :  K 2Mn 208-f 
ioFeS04.  7  H20  +  8  H2S04  =  K2S04  +  2  MnS04  + 
5  Fe2(S04)3+  I5H20.  The  standard  solutions  can  be 
made  any  suitable  strengths,  but  as  we  have  already  in 
use  a  solution  of  permanganate,  3-95  grm.  to  1  litre,  it  can 
be  used  for  the  ferrous  sulphate  :  1  c.c.  =  0-03475  grm. 
FeS04  7H20.  Similarly,  standard  ferrous  sulphate  solu- 
tion 3475  grm.  per  litre,  1  c.c.  =  3-95  mgr.  permanganate. 

Carbolic  Acid,  or  phenol,  C6H5OH,  is  detected 
qualitatively :  (1)  By  its  odour  ;  (2)  Add  a  few  drops  of 
ferric  chloride — a  violet  coloration  discharged  by  acetic 
acid  (distinction  from  salicylic  acid)  ;  (3)  Bromine  water 
gives  a  yellowish- white  precipitate  of  tribromophenol, 
even  in  very  dilute  solutions  ;  (4)  Add  one-fourth  volume 
of  ammonia  and  then  a  few  drops  of  dilute  bleacbing- 
powder  solution — a  blue  colour  develops. 

Quantitatively,  carbolic  acid  is  estimated  by  the  process 
of  Koppeschaar.  The  phenol  is  precipitated  as  tri-bromo- 
phenol  by  the  addition  of  excess  of  bromine  solution.  The 
overplus  of  bromine  is  determined  by  adding  potassium 
iodide  from  which  the  bromine  displaces  iodine,  and  the 
amount  of  the  latter  is  found  by  titration  with  N/10 
sodium  thiosulphate  solution. 

C6H5-OH  +  3Br2  «  C6H2Br3-OH  +  3HBr. 

Br2  +  2KI  =  2KBr  +  I2. 

2Na2S203  -f  It»  Na2S406  +  2NaI. 

The  bromine  solution  used  may  be  N/10,  or  can  have  its 
strength  estimated  at  the  time  in  terms  of  iodine  and 
sodium  thiosulphate. 

Process.— Weigh  out  1-556  grm.  of  the  sample,  and 
dissolve  in  sufficient  water  to  make  1000  c.c.  Take  25  c.c. 
of  dilution  (==  0*0389  grm.  of  specimen)  in  a  glass-stop- 
pered bottle  ;  add  30  c.c.  N/10  bromine  solution  ;  5  c.c.  of 
strong  hydrochloric  acid ;  and  5  c.c.  of  potassium  iodide 
solution  (20  per  cent  weight  in  volume).  Stopper  bottle 
quickly  and  shake  well.  Remove  stopper  and  wash  neck 
and  stopper,  allowing  washings  to  run  into  bottle.  Add 
1  c.c.  of  chloroform  and  shake  mixture.     Titrate  with  N/10 


138  PUBLIC    HEALTH    CHEMISTRY 

sodium  thiosulphate  solution,  and  number  of  c.c.  of  same 
required  subtracted  from  30  gives  number  of  c.c.  of 
N/10  bromine  solution  absorbed.  From  the  quantities 
taken,  this  number,  multiplied  by  four,  gives  percentage 
of  phenol  in  sample. 

The  chief  impurities  in  carbolic  acid  are  the  light  and 
heavy  coal-tar  oils,  which  are  largely  composed  of  hydro- 
carbons of  the  benzene  series.  The  light  oils  float  on  water, 
and  the  heavy  sink;  hence  the  terminology.  In  "  carbolic 
powders  "  there  is  sometimes  very  little  phenol,  it  being 
replaced  by  cresols  or  sulphites.  Even  when  present  the 
phenol  may  be  rendered  inert  by  having  lime  as  a  basis 
instead  of  silica.  Pure  phenol  crystallizes  in  long  colourless 
prisms,  melts  at  420  C,  boils  at  i83°C,  is  soluble  in  water 
(1  part  in  15  at  200  C),  and  is  very  soluble  in  alcohol,  ether, 
benzene,  chloroform,  carbon  disulphide,  and  glacial  acetic 
acid.  Commercial  phenol  is  a  colourless  crystalline  mass, 
which  gradually  acquires  a  reddish  colour,  and  deliquesces 
on  exposure  to  the  air. 

Cresols  are  found  in  coal  tar,  give  a  blue  colour  with 
ferric  chloride,  and  are  otherwise  similar  to  phenol.  They 
are  hydroxy  derivatives  of  toluene  or  methyl-benzene,  and 
are  known  as  ortho-,  meta-,  and  para-cresol,  all  of  them 
being  present  in  coal  tar  and  in  the  tar  from  pine  and 
beech  woods.  Ortho-cresol  melts  at  310  C.  and  boils  at 
1880  C.  ;  meta-cresol  at  40  to  50  C.  and  2010  C.  ;  and 
para-cresol  at  360  C.  and  1980  C,  respectively.  Creosote  is 
obtained  by  distillation  of  wood  tar,  and  contains  among 
other  things  cresols  and  guaiacol. 

Carbolic  Powders. —  A  good  powder  should  contain 
not  less  than  15  per  cent  of  tar  acids  (crude  carbolic  acid), 
of  which  62-5  per  cent  is  crystallizable  at  150  to  200  C. 
when  examined  by  C.  Low's  test.  The  base  should 
contain  no  lime  or  chalk.  Test  reaction  of  powder  with 
litmus.  If  alkaline,  it  contains  free  lime.  If  otherwise, 
the  base  is  probably  siliceous. 

Examination. — 

I.  If  the  base  is  silica.  Mix  the  powder  well  and  take 
50  grm.  Extract  with  150  c.c.  of  methylated  spirit, 
which  dissolves  all  the  tar  acids  not  in  combination  with 
lime.     Now  separate,  and  mix  extract  with  50  c.c.  of  10  per 


DISINFECTANTS,     ETC.  139 

cent  KOH  or  NaOH.  Distil  off  spirit  and  evaporate  to 
about  30  c.c.,  and  cool.  If  any  tar  oils  separate  out,  filter 
them  off,  run  the  filtrate  into  a  burette,  and  add  cautiously 
and  a  little  at  a  time,  50  per  cent  sulphuric  acid,  until  the 
alkali  present  is  completely  neutralized.  The  tar  acids 
are  liberated,  and  will  rise  to  the  surface  and  form  a  separate 
layer,  the  volume  of  which  can  be  read  off.  As  the  sp.  gr. 
of  crude  carbolic  acid  is  about  1050,  the  volume  x  1-05 
will  give  the  weight,  and  the  result  x  2  gives  the  percentage 
of  available  carbolic  acid  present  in  the  powder. 

2.  This  method  extracts  all  the  carbolic  present,  and  is 
suitable  for  samples  which  contain  free  lime.  50  grm. 
of  the  previously  well-mixed  powder  are  placed  in  a  large 
mortar,  and  a  cold  mixture  of  equal  parts  of  sulphuric 
acid  and  water  (i.e.,  50  per  cent)  is  added  drop  by  drop, 
with  constant  stirring,  until  all  the  lime  has  been  converted 
into  sulphate,  and  the  mixture  is  just  acid.  This  is 
determined  by  repeated  testing  with  litmus,  removing  and 
moistening  a  small  fragment  of  the  powder.  The  process 
must  not  be  hurried,  or  heat  sufficient  to  volatilize  the 
free  phenol  present  will  be  generated  (takes  one  hour). 
The  result  is  a  dry  powder,  free  from  lumps.  If  it  is 
moist,  add  some  calcium  sulphate.  Now  extract  with  four 
lots  of  ether,  filtering  each  time  the  supernatant  ether 
through  a  small  filter  into  50  c.c.  of  10  per  cent  NaOH. 
Agitate  well  and  distil  off  the  ether  (almost).  Transfer  to 
a  separator,  washing  out  flask  with  small  quantities  of 
water  and  ether,  which  are  added  to  the  same.  Separate, 
wash  ethereal  layer  with  water,  separate  again,  and  mix 
with  first  lot.  Reduce  bulk  by  evaporation  to  30  c.c, 
transfer  to  a  burette,  and  treat  as  in  (1). 

Sodium  Sulphite  and  Sulphurous  Acid. — See  page 
121. 

Zinc  Chloride. — Estimate  gravimetrically  by  precipita- 
tion with  Am  2S,  filter,  dry,  ignite,  and  weigh  as  ZnO. 

Copper  Sulphate. — Precipitate  as  CuO  with  NaOH. 
Collect,  dry,  ignite,  and  weigh  as  CuO. 


The   "  Lancet "    Commission   appointed  to    inquire   into 
the  Standardization  of  Disinfectants,  issued  a  report,  which 


140  PUBLIC    HEALTH    CHEMISTRY 

is  published  in  the  "  Lancet "  for  1909,  Vol.  ii.,  pages  1454 
(Chemical),  1516  (Bacteriological),  and  1612  (Summary  and 
Conclusions) . 

The  chemical  part  is  here  dealt  with.  Disinfectant  is 
taken  to  mean  a  substance  capable  of  destroying  disease 
germs,  or  a  "  germicide,"  which  is  a  preferable  word. 

Chemical   Analysis. 

Arguing  that  disinfection  is  a  chemical  process,  the 
standards  laid  down  are  these  : — 

1.  A  standardized  disinfectant  should  contain  a  certain 
proportion  of  an  accredited  germicidal  substance  ;    and 

2.  If  it  is  presented  as  a  soapy  mixture,  which  makes 
an  emulsion  with  water,  such  dilution  in  water  should 
show  active  Brownian  movements  of  the  particles 
distributed  in  the  mixture. 

The  coal-tar  disinfectants  form  the  majority  of  those 
sold  to  the  public.  They  consist  of  varying  quantities  of 
phenolic  bodies,  with  inert  tar  oils,  and  in  many  cases 
soap  and  resins  or  other  emulsifying  agents,  such  as  gelatin 
or  dextrin,  etc.  The  analysis  of  these  mixtures  necessitates 
the  separation  of  the  soap,  resin,  or  oily  hydrocarbons 
present,  before  the  presence  of  phenol,  cresol,  or  other 
phenoloid  body  can  be  properly  tested  for.  The  existing 
methods  were  found  to  be  inconvenient,  tedious,  and 
troublesome,  and  the  following  method  was  devised  and 
worked  out  by  testing  a  large  series  of  these  mixtures. 
Distillation  methods  were  avoided,  because  of  the  relatively 
large  quantity  of  disinfectant  required,  and  the  incon- 
venience of  the  operations  in  any  but  a  technical 
laboratory,  while  solvent  processes  were  objected  to,  on 
account  of  the  difficulty  in  separating  the  solvent,  and 
also  because  no  differentiation  of  the  kind  of  phenol 
present  was  attempted. 

The    Lancet- Acetone-Baryta    (L.A.B.)     Method. — 

1.  For  fluids  containing  soaps  and  resins  as  emulsifiers. 
The  process  consists  in  making  an  emulsion  with  a  known 
weight  of  the  disinfectant  mixed  with  water  ;  precipitating 
the  soaps  and  (or)  resins  by  adding  a  strong  solution  of 


DISINFECTANTS,    ETC.  141 

baryta  ;  filtering,  when  all  the  phenols  are  in  the  filtrate, 
and  are  estimated  as  later  described.  The  residue  on  the 
filter  contains  the  soaps  and  resins  as  barium  compounds, 
and  the  neutral  oils.  On  shaking  it  up  with  acetone,  the 
neutral  oils  are  dissolved  out,  and  on  nitration  again,  the 
residue  left,  if  treated  with  HC1,  yields  free  fatty  acids 
and  resins,  which  can  be  dissolved  out  with  ether,  the 
ether  evaporated,  and  the  residue  weighed.  The  acetone 
filtrate  containing  the  neutral  oils  can  be  examined  for 
these. 

Process. — Take  10  grm.  of  disinfecting  fluid,  in  a 
300  c.c.  Erlenmeyer  flask.  Add  100  c.c.  of  distilled  water, 
and  shake  well.  Now  add  15  grm.  of  barium  hydrate 
crystals,  attach  flask  to  a  reflux  condenser,  and  digest  at 
ioo°  C.  for  half  an  hour  by  immersion  in  a  water-bath, 
shaking  at  intervals.  Cool,  and  while  cooling  spread  out 
the  soaps  and  resins  with  a  glass  rod.  Decant  off  clear 
liquid  (baryta  solution),  or  filter  through  an  asbestos  plug 
by  aid  of  water  suction.  In  either  case,  wash  out  flask 
with  warm  baryta  solution,  and  decant  after  settling,  or  filter 
into  previous  nitrate,  making  up  bulk  to  250  or  300  c.c. 
exactly.  Of  the  filtrate,  50  c.c.  are  taken  in  a  separator, 
HC1  is  added  till  acid,  and  then  some  CaCl  2.  The  liberated 
phenols  are  extracted  with  ether.  On  separation  the  ether  is 
evaporated  by  putting  the  fluid  in  a  tall  glass  flask,  which 
is  put  on  a  hot  plate  at  370  C,  and  evaporation  encouraged 
by  blowing  a  current  of  air  into  the  flask.  The  residue 
is  in  this  way  also  made  water-free,  and  on  cooling  is 
weighed  in  the  flask.  The  weight  of  the  flask  is  deducted, 
and  the  remainder  is  the  weight  of  phenol  bodies  present. 
This  is  tested,  as  if  it  were  pure  phenol,  by  the  bromine 
absorption  process.  The  residue  is  dissolved  in  NaOH, 
excess  of  bromine  solution  in  NaOH  added,  and  then  HC1. 
The  phenol  present  absorbs  bromine.  The  amount  of  un- 
used Br  is  measured  by  adding  KI,  and  titrating  the  iodine 
liberated  in  place  of  bromine,  with  N/10  sodium  thio- 
sulphate  solution.  The  difference  from  the  amount  of 
bromine  added  (determined  by  a  blank  experiment)-  is  the 
amount  of  bromine  absorbed.  This  multiplied  by  the  figure 
0-195,  gives  the  equivalent  amount  of  carbolic  acid  present. 
Any  notable  difference  of  this  amount  from  the  weight  of 


142  PUBLIC    HEALTH    CHEMISTRY 

the    residue,    shows    that    the   phenoloid    present    is  not 
phenol  (C6H5  OH). 

C6H5.OH  +  3-  Br  2  =  C6H2Br3.OH  +  3.HBr. 
94  480 

The  water  present  may  be  obtained  by  adding  to 
25  grm.  of  the  fluid,  exactly  10  c.c.  of  10  per  cent  sulphuric 
acid,  and  then  25  c.c.  of  petroleum  (white  spirit).  Shake 
well,  and  allow  to  settle.  The  clear  under  liquid  is 
accurately  measured  in  a  narrow  graduated  cylinder,  and 
the  increase  above  10  c.c.  is  taken  as  amount  of  water  in 
25  grm.  of  disinfecting  fluid. 

The  analyses  undertaken  showed  a  range  of  5  to  66  per 
cent  in  phenolic  bodies,  and  39  to  94  per  cent  in  inert  bodies 
such  as  soap,  resin,  neutral  oils,  alkalies,  and  water. 

2.  For  fluids  containing  neither  soap  nor  resin  as 
emulsifiers.  There  are  preparations  in  which  gelatin  (in 
izal)  and  dextrin  (in  okol)  are  used  as  emulsifiers.  In  these 
the  use  of  baryta  is  omitted,  but  the  phenoloids  are 
dissolved  right  away  in  excess  of  absolute  alcohol  or 
acetone,  which  also  precipitates  the  gelatin  or  dextrin. 
Any  neutral  oils  present  are  in  this  case  along  with  the 
phenoloids,  so  that  the  residue  left  is  the  dextrin  or 
gelatin.  The  neutral  oils  are  got  by  adding  to  the  solution 
in  acetone,  10  per  cent  NaOH,  and  diluting  freely.  They 
may  be  dissolved  out  by  addition  of  20  c.c.  of  petroleum 
spirit.  From  the  filtrate,  the  phenoloids  are  estimated 
as  before. 

Summary  and    Conclusions. 

On  tabulating  the  results  along  with  the  carbolic  acid 
co-efficients  found  in  the  bacteriological  investigation 
it  was  seen  that  the  wider  the  difference  between  the 
percentage  of  phenoloids  present  and  the  equivalent  of 
these  in  carbolic  acid  (calculated  from  the  bromine 
absorption  of  the  phenoloids),  the  higher  was  the  car- 
bolic acid  co-efficient  of  the  disinfectant  for  B.  Coli. 
In  fact,  the  differences  between  these  two  figures,  that 
is  the  percentage  of  phenoloids  and  the  calculated 
equivalent  of  this  in  carbolic  acid,  formed  a  series 
running  parallel  with  that  of  the  co-efficients,  with  few 


DISINFECTANTS,     ETC.  143 

•exceptions.  When  these  differences  were  all  divided  by  3, 
the  series  of  numbers  were  in  many  cases  identical  with, 
or  very  close  to,  the  carbolic  acid  co-efficient  independently 
arrived  at  for  each  substance.  In  the  cases  where  there 
was  a  divergence,  the  disinfectant  did  not  form  an  emulsion 
with  water,  and  the  solution  did  not  exhibit  Brownian 
movements.  Thus,  it  was  deduced  that  the  formula 
(P-B)  4-3  give's  the  carbolic  acid  co-efficient  for  B.  coli 
of  the  disinfectant  tested,  where  P  is  the  percentage  of 
phenoloids  present,  B  the  carbolic  acid  equivalent  of 
these  (calculated),  and  3  an  arbitrary  constant. 

From  a  chemico-physical  point  of  view,  it  is  concluded 
that  for  tar  disinfectants,  at  any  rate,  they  should  contain 
a  reasonable  quantity  of  active  bodies,  phenols  or 
phenoloids,  and  that  the  dilution  of  them  in  water  should 
show  active  Brownian  movements,  that  is,  should  form  a 
satisfactory  emulsion.  The  formula  (P  —  B)  4-3  gives  a 
ready  means  of  estimating  the  carbolic  co-efficient  of  a 
disinfectant,  and  its  value  should  be  at  least  over  unity, 
that  is,  be  equal  to  pure  carbolic  acid.  A  number  of  the 
disinfectants  at  present  in  common  use  give  values  of 
5  to  9  by  this  method. 


CHAPTER     VIII. 


CHEMICAL     APPENDIX. 


Table  of  Glaisher's  Factors. 


Reading  of 

Reading  of 

Reading  of 

Reading  of 

the  dry-bulb 
therm. 

Factor 

the  dry-bulb 
therm. 

Factor 

the  dry-bulb 
therm. 

Factor 

the  dry -bulb 
therm. 

Factor 

F. 

F. 

F, 

F. 

IO° 

878 

33° 

3-01 

56° 

1-94 

79° 

1-69 

11° 

878 

34° 

2-77 

57° 

1 

92 

8o° 

i-68 

12° 

878 

35° 

2-60 

58° 

1 

90 

8i° 

i-68 

13° 

877 

36° 

2-50 

59° 

1 

89 

820 

1-67 

14° 

876 

37° 

2-42 

6o° 

1 

88 

83° 

1-67 

15° 

8-75 

38° 

2-36 

6i° 

1 

87 

84° 

1-66 

1 6° 

870 

39° 

2-32 

62° 

1 

86 

85° 

1-65 

17° 

8-62 

4°° 

2-29 

63° 

I 

85 

86° 

1-65 

i8° 

8-50 

4i° 

2-26 

64° 

1 

83 

87° 

1-64 

*9° 

8-34 

42° 

2-23 

65° 

1 

82 

88° 

1-64 

20° 

8-14 

43° 

2-20 

66° 

1 

81 

89° 

1-63 

21° 

7-88 

44° 

2-1.8 

67° 

1 

80 

9o°    . 

1-63 

22° 

7-60 

45° 

2-16 

68° 

1 

79 

9i° 

1-62 

23° 

7-28 

46° 

2-14 

69° 

1 

78 

92° 

1-62 

24° 

6-92 

47° 

2-12 

70° 

1 

77 

93° 

i-6i 

25° 

6-53 

48° 

2-IO 

7i° 

1 

76 

94° 

i-6o 

26° 

6-o8 

49° 

2-08 

72° 

1 

75 

95° 

i-6o 

27° 

5-6i 

5o° 

2-06 

73° 

1 

74 

96° 

1-59 

28° 

5-12 

5i° 

2-04 

74° 

1 

73 

97° 

1-59 

29° 

4'63 

52° 

2-02 

75° 

1 

72 

98° 

1-58 

3o° 

4-15 

53° 

2-00 

76° 

1 

7i 

99° 

1-58 

3i° 

3-60 

54° 

1-98 

77° 

1 

70 

IOO° 

i-57 

32° 

3'32 

55° 

1-96 

78° 

1*69 

Short  Alcohol  Table. 


Specific 

Volumes 

Specific 

Volumes 

Specific 

Volumes 

gravity 
at  6o°  F. 

per  cent  of 

gravity 
at  6o°  F. 

per  cent  of 

gravity 
at  6o°  F. 

per  cent  of 

alcohol 

alcohol 

alcohol 

IOOO-O 

O-OO 

990-2 

7-00 

979.O 

17-00 

999.9 

0-05 

989-0 

8-00 

978-0 

18-00 

999'8 

0-15 

987-8 

9-00 

977-0 

I9-00 

999-1 

°-55 

986-6 

IO'OO 

976-0 

20-00 

998-5 

I'OO 

985-4 

ii-oo 

970-9 

25-00 

997.0 

2-00 

984-3 

12-00 

965-4 

30-00 

995-6. 

3-00 

983-2 

I3-00 

959-2 

35-oo 

994-2 

4-00 

982-I 

I4-00 

951-9 

40-00 

992-9 

5-00 

981-1 

I5-00 

991-5 

6-oo 

980-0 

16-00 

CHEMICAL    APPENDIX 


145 


Table  showing  the  Weight  of  i  Cubic  Foot  of  Water  Vapour. 


Weight  in 

Weight  in 

Weight  in 

Weight  in 

Temp. 

grains  ot  a 

Temp. 

grains  of  a 

Temp. 

grains  of  a 

Temp. 

grains  of  a 

F. 

cubic  foot 

F. 

cubic  foot 

F. 

cubic  foot 

F. 

cubic  foot 

of  vapour 

of  vapour 

of vapour 

of  vapour 

o° 

o-55 

26° 

1-68 

51° 

4-24 

760 

9-69 

1° 

o-57 

27° 

i-75 

52° 

4'39 

77° 

9-99 

2° 

o-59 

28° 

1-82 

53° 

4'55 

780 

10-31 

3° 

0-62 

290 

1-89 

54° 

4-71 

79° 

10-64 

4° 

0-65 

300 

1-97 

55° 

4-87 

8o° 

10-98 

5° 

o-68 

3i° 

2-05 

56° 

5*04 

8i° 

11-32 

6° 

0-71 

32° 

2-13 

57° 

5'2i 

82° 

11-67 

7° 

074 

33° 

2-21 

58° 

5*39 

83° 

12-03 

8° 

077 

34° 

2-30 

59° 

5-58 

84° 

12-40 

9° 

0-80 

35° 

2'39 

6o° 

5'77 

85° 

12-78 

IO° 

0-84 

36° 

2-48 

6i° 

5-97 

86° 

13-17 

n° 

o-88 

37° 

2-57 

62° 

6-17 

87° 

13-57 

12° 

0-92 

38° 

2-66 

63° 

6-38 

88° 

13-98 

13° 

0-96 

39° 

2-76 

640 

6-59 

89° 

14-41 

14° 

I'OO 

40° 

2-86 

65° 

6-8i 

900 

I4-85 

15° 

1-04 

4i° 

2-97 

66° 

7.04 

91° 

15-29 

16° 

1-09 

42° 

3-08 

67° 

7*27 

92° 

15*74 

i7° 

1-14 

43° 

3-20 

68° 

7'5i 

93° 

16-21 

i8° 

1-19 

44° 

3*32 

69° 

7-76 

94° 

16-69 

i9° 

1-24 

45° 

3'44 

700 

8-oi 

95° 

17-18 

20° 

1-30 

46° 

3-56 

7i° 

8-27 

96° 

17-68 

21° 

1-36 

47° 

3'69 

72° 

8-54 

97° 

18-20 

22° 

1-42 

48° 

3-82 

73° 

8-82 

98° 

18-73 

23° 

1-48 

49° 

3-96 

74° 

9-10 

99° 

19-28 

24° 

i'54 

500 

4-10 

75° 

9-39 

IOO° 

19-84 

25° 

i-6i 

Table   for  Ascertaining  the   Spirit  Value   of 
Acetic   Acid  in  Beer. 


Per- 
centage 

Corresponding  degrees  of  spirit  indication. 

acid 

o'oo 

o'oi 

0-02 

0*03 

0*04 

005 

0*06 

0*07 

0*08 

0*09 

O-O 

— 

0-02 

0-04 

0-06 

0-07 

0-08 

0-09 

O-II 

0-I2 

013 

O-I 

OT4 

0-15 

0-I7 

0-18 

0-19 

0-21 

0-22 

0-23 

0-24 

0-26 

0-2 

0-27 

0-28 

0-29 

0-31 

0-32 

o-33 

o-34 

o-35 

o-37 

0-38 

0-3 

0-39 

0-40 

0-42 

o-43 

0-44 

0-46 

0-47 

0-48 

0-49 

0-51 

0-4 

0-52 

o-53 

o-55 

0-56 

o*57 

o-59 

0-60 

o-6i 

0-62 

0-64 

o-5 

0-65 

o-66 

0-67 

0-69 

0-70 

0-71 

0-72 

o-73 

o-75 

0-76 

o-6 

0-77 

0-78 

0-80 

o-8i 

0-82 

0-84 

0-85 

o-86 

0-87 

0-89 

10 


146 


PUBLIC    HEALTH    CHEMISTRY 


Table    showing    Degrees    of    Spirit    Indication 
with  corresponding  degrees  of  gravity  lost. 


Spirit 
Indication 


Degrees 
and 
tenths 


4-o 
•i 

•2 

•3 
•4 
•5 
•6 

•7 
•8 

•9 
•i 

•2 

•3 
'4 
•5 
•6 

•7 
•8 

•9 

6-o 

•i 

•2 

*3 
•4 
•5 
•6 

•7 


Hundredths  of  a  degree. 


o'oo       o'oi        o*02        0*03        0*04       0*05        006        0*07        o'o8       o  OQ 


I5-IO 
I5-50 

16-00 
16-40 
16-80 
17.30 
17-70 
18-20 
18-60 
19-10 
19-50 
19-90 
20-40 
20-90 
21-30 
21-80 

22-20 
22-70 
23-IO 
23-60 
24-IO 
24-60 
25-00 
25-50 
26-00 
26-40 
26-90 
27-40 
27-80 
28-30 


15-14 

15-55 
16-04 
16-44 
16-85 
17-34 
17-75 
18-24 
18-65 
19-14 
19-54 
19-95 
20-45 
20-94 

21-35 
21-84 

22-25 

22-74 

23-15 
23-65 
24-15 
24-65 

25-05 

25-55 
26-04 

26-45 
26-95 
27-44 
27-85 
28-35 


15-18 
15-60 
16-08 
16-48 
16-90 
17-38 
17-80 
18-28 
18-70 
19-18 
19-58 

20-00 

20-50 

20-98 

2I-40 

21- 

22-30 

22-78 

23-20 

23-70 

24-20 

24-68 

25-IO 

25-60 

26-08 

26-50 

27-00 

27.48 

27-90 

28-40 


15-22 
15-65 

16-12 

16-52 

16-95 
17-42 
17-85 
18-32 

i8-75 
19-22 
19-62 

20-05 
20-55 

21-02 

21-45 
21-92 

22-35 

22-82 

23-25 
23-75 
24-25 
24-72 
25-15 
25-65 

26-12 
26-55 
27-05 
27-52 
27-95 
28-45 


15-26 
15-70 
16-16 
16-56 
17-00 
17.46 
17.90 
18-36 
i8-8o 
19-26 
19-66 

20-10 

20-60 
21-06 

21-50 

21-96 
22-40 

22-86 

23-30 
23-80 

24-30 
24-76 
25-20 
25-70 

26-16 
26-60 
27-10 

27-56 

28-00 

28-50 


15-30 

15-75 
16-20 
i6-6o 

I7-05 
17.50 

17-95 
18-40 
18-85 
19-30 
19.70 
20-15 
20-65 

2I-IO 

21-55 

22-00 

22-45 

22-90 

23-35 
23-85 
24-35 
24-80 

25-25 
25-75 
26-20 
26-65 
27-15 
27-60 
28-05 
28-55 


15-34 
15-80 
16-24 
16-64 
17-10 

17-54 
i8-oo 
18-44 

18-90 

19-34 
19-74 

20-20 
20-70 
21-14 

21-60 
22-04 
22-50 

22-94 

23.40 
23.90 
24.40 
24.84 
25-30 
25-80 

26-24 
26-70 
27-20 

27-64 
28-10 
28-60 


15-38 
15-85 

16-28 

16-68 

I7-I5 
17-58 
18-05 
18-48 
18-95 
19-38 
19-78 
20-25 

20'75|20 
2I-l8|2I 
2I-65|2I 
22-o8|22 
22-55122 
22-98  23 


•42  15.46 
•90  15-95 
•32  16-36 
72  16-76 
20  17-25 
62  17-66 
IO  I8-I5 
.  I8-56 
OO  19*05 
19-46 

19-86 


23-45 
23*95 
24-45 
24-88124 

25*35  25 
25-85  25 
26-28  26 
26-75  26 
27-2527 
27-68  27 
28-1528 
28-65  28 


20-35 
20-85 
21-26 
21-75 

22-16 
22-65 
23-06 
23-55 
24-05 
24*55 
24-96 
25-45 

25-95 
26-36 
26-85 

27*35 
27-76 
28-25 

28-75 


CHEMICAL    APPENDIX 


147 


Table  showing  Degrees  of  Spirit  Indication 

WITH  CORRESPONDING    DEGREES    OF    GRAVITY   LOST    (continued). 


Spirit 
indication 


Degrees 
and 
tenths 


7-o 
•i 

•2 

•3 
•4 
•5 
•6 

•7 
•8 

•9 

8-o 

•i 

•2 

•3 
•4 
•5 
•6 

•7 
♦8 

•9 

9-o 

•I 

•2 

•3 
'4 
•5 
•6 

•7 
•8 

•9 


Hundredths  of  a  degree. 


28-8o 
29-20 
29-70 
30-20 
30-70 
31-20 
31-70 
32-20 
32-70 
33-2o 
33-7° 
34-30 
34-80 

35-4° 
35-90 
36-50 
37-00 
37-5o 
38-00 
38-60 
39-10 
39.70 
40-20 
40-70 
41-20 
41-70 
42-20 
42-70 
43-20 
43-7o 


O'OI  0'02  0*03  0'04 


28-84 


29-30 
29-80 

30-30 
75|30-80 
•253I.3O 


3I-80 
32-30 
32-80 

33-3° 
33-82 
35J34-40 
86J34-92 
'45I35-50 
■96'36-02 
36-60 


37-10 
37-60 
38-12 
38-70 
39-22 

•75  39-8o 

25 

•75 

25 

•75 

25 

•75 

•25 

75 


40-30 
40-80 

4I-3° 
41-80 
42-30 
42-80 

43-3° 
43-80 


28-92 
29-35 
29-85 
3°-35 
30-85 
31-35 
31-85 
32-35 
32-85 
33-35 
33-88 
34-45 
34-98 
35-55 
36-08 
36-65 
37-15 
37-65 
38-18 

38-75 

39-2 

39-85 

40-35 

40-85 

4X'35 
41-85 
42-35 
42-85 
43*35 
43-85 


29-00 
29-45 
29*95 
3°-45 
3°-95 
31-45 
31-95 
32-45 
32-95 
33-45 
34-00 

34-55 
35-1° 
35-65 
36-20 

36-75 
37-25 
37-75 
38-30 
38-85 
39-4° 
39-95 
4°-45 
4°-95 
41-45 
4J-95 
42-45 
42-95 


"4°  43*45 
•90  43-95 


o"o6       0*07       o"o8        0*09 


29-08 

29-55 

30-05 
3°-55 
3I-05 

31-55 
32-05 

32-55 
33-o5 
33-55 
34-12 

34-65 
35-22 

35-75 
36-32 
36-85 
37-35 
37-85 
38-42 
38-95 
39-52 
40-05 

4°-55 
41-05 

4I#55 
42-05 
42-55 
43-°5 
43-55 
44-°5 


29-12 
29-60 
30-10 
30-60 
31-10 
31-60 
32-10 
32-60 

33-1° 
33-6o 
34-i8 
34-7° 
33-28 
35-8o 
36-38 
36-90 
37-4° 
37-9° 
38-48 
39-oo 
39-58 
40-10 
40-60 
41-10 
41-60 
42-10 
42-60 
43-10 
43-60 
44-10 


29-16 
29*65 
30-I5 
30-65 
3i-i5 
31-65 
32-15 
32-65 
33-15 
33-65 
34-24 
34-75 
35-34 
35-85 
36-44 
36-95 
37-45 
3-7-95 
38-54 
39-o5 
39-64 
40,I5 
40-65 
4*'I5 
41*65 
42-15 
42-65 
43-15 
43-65 
44-15 


Part  II. 
PUBLIC     HEALTH     BACTERIOLOGY. 

BACTERIOLOGY  is  one  of  the  more  recent  sciences, 
and  is  becoming  larger  and  more  complex  every 
year.  It  is*  now  of  extreme  importance  to  the  Hygienist, 
as  it  is  also  to  the  Agriculturalist,  and  in  various  Arts 
and  Industries.  A  short  glance  at  its  evolution  will  serve 
to  make  the  methods  and  processes  to  be  hereinafter 
studied  more  intelligible. 

Leeuwenhoek,  a  native  of  Delft,  in  Holland,  produced 
the  first  really  good  microscope  and,  on  examining  with  it 
the  contents  of  the  intestinal  canal  in  horses,  frogs,  pigeons, 
and  fowls,  and  his  own  diarrhoea  stools,  he  saw  small  moving 
and  living  forms.  This  was  in  1675,  and  eight  years  later 
he  examined  tartar  scraped  from  teeth,  and  described 
and  depicted  minute  organisms  such  as  we  recognize  at 
the  present  day.  These  discoveries  accorded  with  various 
theories  which  were  prevalent  at  the  time,  and  a  micro- 
organismal  basis  for  disease  became  widely  accepted, 
which,  though  much  the  same  as  prevails  now,  was  built 
on  very  slender  foundations.  Plenciz  thereafter  insisted 
on  the  specific  character  of  the  contagious  diseases,  and 
explained  the  incubation  period  as  dependent  on  the 
growth  of  a  germ  in  the  body,  which  had  not  yet  made  its 
presence  manifest.  Muller  systematized  the  morphology. 
Then  the  theory  of  spontaneous  generation  or  abiogenesis 
arose.  Dr.  Needham  boiled  a  beef  infusion,  kept  it  in  a 
well-stoppered  bottle,  and  found  that,  on  keeping,  it  putre- 
fied. He  argued  that  the  boiling  killed  all  the  organisms 
in  the  infusion ,  and  that  therefore  the  putrefaction  of  the 
infusion  pointed  to  the  existence  of  a  special  vegetative 
force  which  produced  fresh  organisms.  Spallanzani  worked 
at  the  subject,  and  found  that  on  boiling  for  one  hour  and 
then  hermetically  sealing,  no  putrefaction  ensued.  It  was 
objected  that  the  air  was  shut  from  the  vessel,  and  that 
this  might  be  necessary.  This  objection  was  met  by 
Schulze  in  1836  by  fitting  a  flask  with  right-angled  tubes, 


PUBLIC    HEALTH    BACTERIOLOGY         149 

and  filtering  all  the  air  through  sulphuric  acid  in  the  one 
and  potash  in  the  other.  Schroeder  and  von  Dusch  found 
(in  1854)  that  filtration  through  cotton-wool  sufficed, 
and  then  Hoffmann,  Chevreul,  .and  Pasteur  showed  that 
it  was  quite  sufficient  to  draw  out  the  neck  of  the  bottle 
and  bend  it  downwards ;  and  they  argued  that  germs 
obeyed  the  laws  of  gravitation  in  the  absence  of  wind. 
Pasteur  demonstrated  that  there  was  a  causal  relation 
between  certain  lowly  organized  parasitic  organisms  and 
certain  diseases  of  animals  and  insects.  His  conclusions 
were  attacked  on  two  grounds  :  (1)  that  these  organisms 
were  not  the  cause  of  disease  at  all,  and  (2)  that  the  germs 
were  not  specific,  i.e.,  one  for  each  fermentation  or  disease. 
The  second  objection  was  a  forcible  one  because,  so  far,  he 
had  not  been  working  with  pure  cultures.  The  first  one  was 
met  by  the  researches  of  Lemaire,  who  proved  that  carbolic 
acid  was  hurtful  to  the  life  of  the  higher  animals  and  plants, 
and  that  the  addition  of  a  small  quantity  of  it  to  fluids 
prevented  the  incidence  of  putrefaction  and  fermentation 
but  did  not  retard  the  action  of  diastase  or  synaptase.  He 
applied  the  same  reasoning  to  the  treatment  of  wounds, 
and  reduced  pus  to  a  minimum  and  got  rapid  healing, 
which  results  he  attributed  to  the  destruction  of  the 
microzoa  and  infusoria  by  the  carbolic  acid  lotions.  Lister 
saw  the  great  importance  of  Pasteur's  work,  and  on  it  and 
independent  research  he  built  up  against  considerable 
opposition  the  theory  and  practice  of  antiseptic  surgery. 
Pathogenic  bacteria  were  also  being  studied,  beginning 
with  Bacillus  anthracis,  which  was  first  observed  by 
Pollender  in  1849,  and  described  by  Davaine  and  Rayer 
in  1850  as  motionless,  thread-like  organisms  and  rods, 
found  in  the  blood  taken  from  animals  affected  with 
splenic  fever.  In  1863  Davaine  suggested  that  these  rods 
were  the  actual  and  specific  cause  of  the  disease,  and  in 
1864  he  demonstrated  (not  rigorously)  that  malignant 
pustule  and  splenic  fever  were  forms  of  one  infection. 
Confusion  was  introduced  by  the  revival  of  the  theory  of 
polymorphism  in  the  form  that  all  contagia  and  miasmata 
are  the  products  of  fungi  or  algae,  and  on  account  of  their 
small  size  are  able  to  pass  through  the  fine  capillary  vessels, 
and    that  when    a    micrococcus  was  found  it  was  only 


150         PUBLIC    HEALTH    BACTERIOLOGY 

necessary  to  trace  it  back  to  some  parent  fungus  or  mould. 
This  not  improbable  theory  was,  even  in  its  crude  form, 
not  easily  met,  as  at  that  time  no  one  was  working  with 
pure  cultures.  Klebs  in  1872  described  in  pus,  rod-like 
bodies  and  "  microspores,"  grouped  in  short  chains  or  in 
longer  threads ;  and  he  filtered  the  pus  through  baked  clay 
cylinders,  and  found  that  the  filtrate  on  injection  into  the 
blood  or  under  the  skin  gave  constitutional  symptoms,  but 
did  not  induce  suppuration  nor  cause  death  ;  though  if  to 
it  were  added  a  small  quantity  of  the  micro-organisms,  a 
true  pyaemia  was  produced. 

Koch  in  1876  demonstrated  the  specificity  of  the  anthrax 
bacillus  by  making  it  satisfy  the  following  four  conditions, 
commonly  called  Koch's  postulates,  namely  : 

1.  The  anthrax  bacillus  was  invariably  found  in  the 
blood  or  tissues  of  animals  affected  with  the  disease. 

2.  The  bacillus  was  cultivated  in  artificial  media  for  an 
indefinite  number  of  successive  generations. 

3.  The  same  disease  was  produced  by  inoculation  of  a 
susceptible  animal  with  the  last  cultivation. 

4.  In  every  such  inoculated  animal  the  specific  microbe 
was  found,  and  similarly  distributed  as  in  animals  infected 
in  the  ordinary  way. 

To  these  Martin  adds  : — 

5.  The  secondary  infective  agent  or  toxin  separable 
from  the  tissues  in  the  natural  disease,  should  have  similar 
chemical  and  physiological  actions  to  the  products 
obtained  from  a  pure  cultivation  of  the  organism. 

Koch  succeeded  in  doing  this  by  being  able  to  make 
pure  cultures  of  the  anthrax  bacillus  on  the  aqueous  humour 
of  the  eye  of  the  ox,  and  in  this  way  was  able  to  carry 
out  the  further  procedures,  and  to  accept  the  results  as  due 
to  the  single  substance  inserted. 

Numerous  similar  investigations  were  now  made,  and 
served  to  corroborate  the  soundness  of  the  germ  theory  of 
disease. 

The  introduction  of  solid  media  by  Koch  in  1882  paved 
the  way  for  an  enormous  advance  in  bacteriological 
technique,  and  numerous  discoveries  of  specific  organisms 
followed,  or  specificity  wa^i  established  : — Staphylococci  in 
1880-83,    Streptococci    1881-84,    Micrococcus    tetragenus 


GENERAL    PRINCIPLES  151 

1881,  Gonococcus  1879-85,  Pneumobacillus  1883,  Pneumo- 
coccus  1884,  Meningococcus  1885,  Tubercle  bacillus  1882, 
Bacillus  mallei  1882,  B.  typhosus  1880-84,  B-  coli>  l886» 
Klebs-Loeffler  1883-84,  Micrococcus  melitensis  1887,  B. 
enteritidis  1888,  B.  tetanus  1884-89,  B.  pestis  1894,  \B. 
enteritidis  sporogenes  1895,  Cholera  v.  1883,  B.  botulinus, 
B.  paracolon  and  paratyphosus,  and  B.  Morax-Axenfeld, 
all  in  1896. 


CHAPTER     IX. 
GENERAL     PRINCIPLES. 
BACTERIOLOGICAL     MEDIA 

may  be  thus  classified  : — 

Nutrient  Broth,  standardized. 

Derivatives  :  Glucose  broth,  lactose  broth,  nutrient 
gelatin,  nutrient  agar-agar,  glycerin  agar,  glucose 
agar,  lactose  agar. 

Peptone  Water. 

Derivatives :      Glucose     peptone     water,     lactose 

peptone    water,    sucrose    and   mannite    peptone 

water. 
MacConkey's  Media. —  Bile-salt  litmus  glucose  peptone 

water ;  bile-salt  neutral-red  lactose  agar. 
Other  Media. — Milk,  potato,  blood  serum,  ascitic  fluid, 

urine,    whey,    gelatin    agar,    beer    wort,    bread, 

eggs ;    nitrate   media,   synthetic   media ;    animal 

tissues,  etc. 

Nutrient  Broth. — 500  grm.  of  lean  beef  finely  minced 
are  steeped  in  one  litre  of  ordinary  water  for  twenty-four 
hours  in  a  cool  place.  The  fat  particles  are  then  skimmed 
off,  the  fluid  strained  off,  and  the  juice  well  pressed  out. 
This  is  then  boiled  for  half  an  hour  to  coagulate  the 
albumins,  filtered,  and  the  bulk  made  up  to  1  litre.  One 
per  cent  of  Witte's  peptones  and  \  per  cent  of  common  salt 
are  then  added  and  dissolved  by  the  aid  of  heat.  The 
broth  is  now  tested  as  to  its  reaction,  and  is  usually  acid. 
Its  acidity  is  determined  by  taking  5  c.c,  diluting  to  50  c.c. 


152         PUBLIC    HEALTH    BACTERIOLOGY 

with  water,  adding  i  c.c.  of  phenolphthalein,  and  titrating 
with  N/io  NaOH.  Boil  one  minute  before  titration. 
Calculate  the  amount  of  N/i  NaOH  required  for  the  litre 
of  broth  made.  The  convention  at  present  in  use  is  to 
leave  the  broth  acid  to  phenolphthalein  to  the  extent  that 
i  c.c.  of  N/i  NaOH  is  required  to  neutralize  ioo  c.c.  of 
broth,  or  10  c.c.  per  litre,  and  the  medium  is  said  to  be 
"  acid  +  10,"  or  +  i  per  cent.  Therefore  add  the  calcu- 
lated amount  of  N/i  NaOH  less  10.  (Some  add  the  full 
amount  of  soda  and  then  make  acid  to  the  desired  extent 
with  N/i  HC1.)  Heat  to  boiling  and  test  again  ;  if  it  needs 
a  further  correction,  boil  again.  Allow  to  cool  to  bring 
down  the  precipitate  of  MgAm  phosphate  caused  by  the 
change  of  reaction.  Filter,  place  in  flasks  or  tubes  (about 
5  c.c),  and  sterilize  in  autoclave  at  1200  C.  for  15  minutes, 
or  at  1300  C.  for  1  minute,  or  for  15  to  60  minutes  on 
three  successive  days  in  a  steam  sterilizer  at  ioo°  C. 

Broth  can  also  be  made  from  Liebig's  Extract  of  Meat, 
using  o*5  per  cent  of  it  instead  of  mince-meat.  The  other 
procedure  is  similar.  Neutralization  can  also  be  effected 
by  adding  saturated  solution  of  NaOH  until  red  litmus  is 
just  turned  blue. 

Meat  Extract  ,or  Fleischwasser  can  be  used  as  a  basis  for 
broth  and  the  media  derived  from  it.  It  is  made  by 
warming  500  grm.  of  minced  beef  or  horseflesh  with 
1  litre  of  water  at  500  C.  for  half  an  hour,  and  then  boiling 
for  half  to  three-quarters  of  an  hour.  Filter,  strain,  make 
up  to  1  litre,  and  then  pour  into  a  flask ;  if  not  to  be  used 
at  once,  sterilize. 

Broth  contains  some  muscle  sugar  or  inosite,  and  to 
get  rid  of  this  is  at  times  inoculated  with  a  young  culture 
of  B.  coli  and  incubated  at  370  C.  for  18  hours,  and  then 
boiled  to  kill  the  organisms. 

Glucose  and  Lactose  are  added  to  sugar-free  broth  (usually 
1  per  cent).  Such  media  are  not  sterilized  in  the  autoclave 
but  in  a  steamer,  because  the  sugars  are  not  stable  at 
high  temperatures. 

Nutrient  Gelatin  is  made  from  broth  by  adding  10  per 
cent  in  winter  and  15  per  cent  in  summer  of  "  gold  label" 
gelatin.  Heat  on  water-bath  (as  little  as  possible)  to 
dissolve  ;   readjust  reaction    (gelatin  makes   the  medium 


GENERAL    PRINCIPLES  153 

acid),  filter,  and  sterilize  in  the  steamer.  If  filtrate  is 
not  clear,  cool  to  6o°  C,  add  whites  of  two  eggs  per  litre, 
re-heat  for  half-an-hour  in  steamer,  and  filter  through 
Chardin  paper  in  warm  filter-jacket. 

Nutrient  Agar. — Add  1*5  per  cent  of  powdered  agar  to 
broth.  Melt  in  the  steamer  at  ioo°  C.  for  1-5  hours. 
Standardize,  and  replace  in  steamer  for  20  minutes  to 
precipitate  phosphates.  Cool  to  6o°  C,  add  two  whites  of 
egg  per  litre,  reheat  for  half  an  hour,  filter  through  Chardin 
paper  by  aid  of  a  hot-water  funnel  or  in  a  steamer,  or  filter 
through  glass-wool.  Tube,  and  sterilize.  Agar  melts 
between  900  and  ioo°  C.  and  remains  fluid  down  to  400  C. 

Glycerin  Agar.  —  Add  6  per  cent  of  glycerin  after 
filtration  ;   tube,  and  sterilize. 

Glucose  and  Lactose  Agar. — To  agar  made  with  sugar- 
free  broth,  add  2  per  cent  of  glucose  or  lactose.  If  to  be 
tinted  with  neutral  red,  add  before  filtration  2  per  cent  of 
a  solution  of  neutral  red  (J  per  cent). 

Litmus  Lactose  Agar. — Add  to  nutrient  agar  prepared 
from  sugar-free  broth  1  to  2  per  cent  of  lactose,  and 
sufficient  litmus  to  give  a  good  colour  (about  5  to  10  c.c. 
of  a  1  per  cent  litmus  solution  per  100  c.c.  of  total  medium). 
Conradi  and  Drigalski's  medhim  is  similar,  plus  nutrose 
and  crystal- violet. 

Peptone  Water, — Dissolve  by  the  aid  of  heat  1  per 
cent  of  peptones  and  J  per  cent  of  NaCl  in  distilled  water. 
Tube,  and  sterilize.  For  water  investigations  it  is  usual 
to  keep  a  stock  solution  ten  times  this  strength. 

Glucose,  lactose,  sucrose,  and  mannite  are  used  with 
peptone  water  plus  Durham's  fermentation  tubes.  Mostly 
in  1  per  cent  strength. 

MacConkey's  Media  are  much  used  in  water  examina- 
tions in  this  country. 

Bile-salt  Litmus  Glucose  Peptone  Water  is  made  in  single 
strength,  and  in  triple  strength  thus  : — Peptone,  20  or 
60  grm.  ;  glucose,  5  or  15  grm.  ;  taurocholate  of  sodium, 
5  or  15  grm. ;  litmus  solution  (10  per  cent  sterile)  100  c.c, 
and  water  to  1  litre.  Put  peptone,  glucose,  bile-salt, 
and  water  in  a  flask  and  heat  in  steamer  for  45  minutes. 
Filter  through  Chardin's  paper,   add  the  filtered  litmus 


154         PUBLIC    HEALTH    BACTERIOLOGY 

solution,  place  in  tubes,  and  put  in  Durham's  fermentation 
tubes.  Steam  for  45  minutes  on  two  successive  days. 
Double  strength  is  also  used.  In  tubing  put  10  c.c. 
of  single  strength,  10  c.c.  of  double,  and  50  c.c.  of  triple, 
into  suitable  tubes.  To  these  are  added  respectively  1  c.c, 
10  c.c,  and  100  c.c.  of  the  water  sample. 

Bile-salt  Neutral-red  Lactose  Agar  is  composed  of  agar 
20  grm.,  peptones  20  grm.,  lactose  10  grm.,  bile-salt 
5  grm.,  neutral-red  aqueous  sterile  solution  (1  per  cent) 
4  c.c,  and  water  to  1  litre.  Dissolve  the  agar,  peptones 
and  bile-salt  in  500  c.c.  of  water  by  heating  in  the  steamer 
for  90  minutes.  Add  rest  of  water,  cool  to  6o°  C,  add  the 
white  of  one  egg,  heat  in  steamer  for  45  minutes,  filter 
through  a  moistened  Chardin  filter-paper  in  a  warm  filter- 
jacket,  heat  filtrate  in  steamer  for  15  minutes,  add  lactose 
and  neutral  red,  put  in  tubes,  and  steam  for  30  minutes 
on  two  successive  days.     The  medium  requires  no  alkali. 

Other  Media. — 

Milk. — Fresh  milk  free  from  preservatives,  with  the 
cream  removed,  and  giving  an  amphoteric  reaction  to 
litmus,  is  poured  into  tubes  and  heated  in  the  steamer  for 
three  successive  days  at  ioo°  C.  (Above  no°  C.  browns 
it.)  To  prove  sterility,  incubate  for  at  least  three  days  at 
370  C.  before  using. 

Litmus  Milk. — Add  sufficient  litmus  to  colour. 

Potato.  —  Scrub  and  wash  a  potato  ;  bore  a  cylinder- 
shaped  piece  ;  split  it  diagonally  and  put  each  half  into  a 
sterile  tube,  with  a  pad  of  wool  at  the  bottom,  and  half  an 
inch  of  aq.  dest.     Plug,  and  sterilize  at  ioo°  C.  on  three  days. 

Blood  Serum.  —  Collect  blood  in  a  sterile  cylinder  and 
allow  to  clot.  Set  aside  for  twenty-four  hours  in  an  ice 
chest.  Pipette  serum  into  tubes  and  place  these  in  a 
sloping  position  in  inspissator  at  750  C.  for  one  hour. 
Repeat  on  two  successive  days.  When  cool,  incubate 
for  twenty-four  hours  at  370  C,  and  if  no  growth,  they  may 
be  considered  sterile. 

Litmus  Whey  (Petruschky's). — Mix  milk  with  equal 
quantity  of  water,  heat  to  400  to  500  C,  and  add  dilute 
HC1  to  precipitate  casein.  Filter,  neutralize  with  NaOH, 
and   heat    for   one   or   two    hours   in  steamer,  filter  till 


GENERAL    PRINCIPLES  155 

clear,  and  if  necessary  neutralize  again.  Add  sterile 
litmus  until  a  violet  hue  is  produced.  Tube,  and  sterilize. 
A  good  medium  for  observing  change  of  reaction. 

STERILIZATION    AND    DISINFECTION. 

i.  Dry  heat : 

(a)  Bright  red  heat  of  flame :  for  platinum  needles. 

(b)  Dull  red  heat  of  flame :   for  knives,  glass  rods, 

etc. 

(c)  Hot  air :  1700  C.  for  1  hour :  for  glass-ware  and 

cotton-wool. 

2.  Moist  heat: 

(a)  Boiling  in  water  at  ioo°  C. :  for  5  minutes  kills 

all  non-sporing  forms:  for  ij  hours  kills 
spores  also. 

(b)  Steam  at  ioo°  C. :  Koch's  steam  sterilizer :   1 J 

hours'  full  steaming,  or  15  minutes'  full 
steaming  on  three  successive  days  :  used  for 
all  media. 

(c)  High-pressure  steam  :   in  autoclave:  atii5°C, 

2  minutes  for  germs,  15  minutes  for  spores. 
Never  used  for  gelatin  media,  or  will  not  re- 
solidify. Never  used  for  carbohydrate 
media,  as  decomposed  into  other  sugars. 

3.  Chemicals:   5   per   cent  carbolic,  o-i    per  cent  per- 

chloride  of  mercury,  etc.     Allow  to  remain  in  con- 
tact for  half-an-hour. 

Discontinuous  sterilization  at  57°-75°  C.  is  used  for 
media,  like  blood  serum,  that  are  changed  at  higher  tem- 
peratures. The  object  is  submitted  to  60  minutes'  heating, 
and  kept  at  200  to  370  C.  until  next  day,  when  the  heating 
is  repeated,  and  the  same  procedure  repeated  for  3  to  8 
days.  This  is  to  cause  spores  present  to  assume  the 
vegetative  form  and  then  to  kill  the  same  on  re-heating. 

CULTURAL     METHODS. 

i.  Inoculation  of  liquid  media,  solid  media,  living  media. 

2.  Isolation  of  pure  cultures  by  (a)  serial  inoculation  ; 

(b)    plate    cultivation    (serial    dilution)  ;     (c)    differential 


156         PUBLIC     HEALTH    BACTERIOLOGY 

sterilization ;  (d)  aerobic  and  anaerobic  cultivation ; 
(e)  deterrent  media  ;  (/)  favouring  media  ;  (g)  inoculation 
into  susceptible  animals. 

3.  Preparation  of  toxins,  vaccines,  and  sera. 

4.  Post-mortem  examination  of  bodies  and  tissues. 

5.  Examination  of  blood,  pus,  sputum,  urine,  cerebro- 
spinal fluid,  exudates,  and  dust,  air,  water,  milk,  sewage, 
soil,  shell-fish,  water-cress,  etc. 

MODES    OF    STUDY. 

Cultures. — Growth  on  or  in  various  media  ;  liquefaction 
of  media  ;  gas  production  ;  acid  or  alkali  production ;  indol 
formation  ;  colour  formation  (pigment)  ;  colour  reduction ; 
proteinchrome  formation ;  sulphuretted  hydrogen  produc- 
tion ;  phosphorescence  ;  nitrate  reduction ;  toxin  formation ; 
ferment  production  and  effects. 

Morphology. — Form,  motility,  flagella,  sporing,  pleo- 
morphism,  colour,  staining  reactions,  capsulated. 

Resistance  to  desiccation,  dry  heat,  moist  heat,  chemical 
agents,  sunlight,  ultra-violet  rays. 

Optimum  Temperature  for  growth,  and  toxin  and  ferment 
formation. 

Pathogenicity  for  (a)  man,  (b)  animals,  (c)  plants. 

Products  of  Growth  in  Host  and  Culture — toxins  soluble 
and  insoluble,  ferments. 

Habitat. 

Immunity. — Mode  of  production  ;  antitoxins,  alexins, 
complement,  phagocytosis,  opsonins,  amboceptors,  anti- 
bodies, agglutinins,  precipitins,  aggressins. 

Anaphylaxis  (from  Gr.  "  against  protection  ") — opposite 
of  Immunity. — A  state  of  excessive  susceptibility  induced  in 
animals  by  the  injection  of  certain  substances  (blood 
serum,  white-of-egg,  milk,  etc.) 

CULTURAL     REACTIONS. 

Inoculate  various  media  and  observe  results  from  day 
to  day  on  incubation  at  200  or  370  C.  (as  directed). 
Label  tubes  with  name  of  organism  and  date  of  inoculation, 
or  mark  with  pencil. 


GENERAL    PRINCIPLES  157 

Broth. — Inoculate  from  agar  cultures  three  broth  tubes, 
one  with  each  of  the  following  bacilli :  B.  fluorescens 
liquefaciens,  B.  subtilis,  and  B.  mycoides.  Incubate  at 
20°  C.  and  examine  in  twenty-four  or  forty-eight  hours. 
Culture  of  B.  fluorescens  liquefaciens,  quite  turbid,  or 
"  universal  turbidity  "  ;  of  B.  subtilis,  quite  clear  but  scum 
is  formed  ;  of  B.  mycoides,  quite  clear  but  deposit  is 
formed. 

Gelatin. — Three  forms  of  culture :  (i)  slant  or  streak, 
(2)  stab,  (3)  shake. 

1.  Slant  or  streak  —  for  non-liquefying  organisms. 
Inoculate  sloped  tubes  with  B.  coli  communis  and  Torula 
alba.  Incubate  at  200  C.  and  examine  after  forty-eight 
hours. 

B.  coli  communis,  spread  over  surface ;  Torula  alba, 
growth  limited  to  line  of  inoculation. 

2.  Stab  culture — to  observe  presence  or  absence  of 
liquefaction.  Inoculate  gelatin  tubes  by  stabbing  with 
B.  mycoides,  B.  megatherium,  and  Vibrio  Finkler-Prior. 
Incubate  at  200  C.  and  examine  after  twenty-four,  forty- 
eight,  and  seventy-two  hours. 

B.  mycoides,  horizontal  liquefaction  ;  B.  megatherium, 
funnel  of  liquefaction  medium  width  ;  V.  Finkler-Prior, 
funnel  of  liquefaction  wide. 

3.  Shake  culture — to  observe  gas  formation.  Melt 
gelatin  at  400  C.  on  water-bath  and  inoculate  as  in  the 
case  of  broth.  Place  in  rack,  and  allow  to  solidify. 
Incubate  for  forty-eight  hours.  Use  B.  coli  communis  and 
B.  subtilis. 

B.  coli  communis,  gelatin  full  of  gas  bubbles ;  B.  subtilis, 
no  gas  formed. 

Agar. — Inoculate  sloped  agar  tubes  with  the  following 
germs,  incubate  at  370  C,  and  examine  after  twenty- 
four  hours. 

Results  should  be  as  follows : — B.  subtilis,  dry  myco- 
derma ;  B.  mycoides,  fine  filaments ;  B.  megatherium, 
confluent  moist  raised  growth ;  B.  proteus,  thin  transparent 
growth  over  whole  surface. 

Potato. — Inoculate,  incubate  at  37°  C,  arid  examine  in. 
twenty-four  hours. 


158         PUBLIC    HEALTH    BACTERIOLOGY 

B.  subtilis,  flesh-coloured  mycoderma  ;  Streptococcus, 
invisible  growth  ;  B.  megatherium,  a  yellow,  raised,  moist 
growth. 

Blood  Serum. — Inoculate,  and  incubate  at  370  C.  for 
two  to  three  days,  and  examine. 

B.  coli  communis,  serum  solid  ;  B.  pyocyaneus,  serum 
liquefied. 

Milk. —  Inoculate,  incubate  at  370  C.  for  forty-eight 
hours,  and  examine. 

B.  coli  communis,  milk  clotted  and  acid  ;  B.  denitri- 
ficans,  no  clot,  alkaline  ;  B.  pyocyaneus,  casein  pre- 
cipitated and  partly  dissolved  ;  Streptococcus,  no  clot, 
-slightly  acid. 

MacConkey's  Broth  with  Durham's  tubes.  Inoculate, 
incubate  at  370  C,  and  examine  daily. 

B.  coli,  acid  and  gas  ;   B.  typhosus,  acid,  no  gas. 

Peptone  Water. — Inoculate,  incubate  at  370  C,  and 
examine  in  four  or  five  days. 

B.  coli  communis,  indol  formed  ;  B.  typhosus,  none  ; 
Sp.  cholerae,  nitroso-indol. 

Aerobic  and  Anaerobic. —  Make  gelatin  stabs  and 
incubate'at  200  C.  for  two  days. 

B.  zopfii,  growth  only  on  surface  (strict  aerobe).  Torula 
alba,  on  surface  and  in  depth  (aerobe  and  facultative 
anaerobe).  B.  butyricus,  growth  only  in  depth  (strict 
anaerobe) . 

Colour    Formation. — 

1.  Inoculate  two  agar  tubes  with  B.  prodigiosus,  and 
incubate  ;  (1)  at  370  C.,  (2)  at  200  C.  Examine  in  forty- 
eight  hours.     (1)  is  white  or  grey,  (2)  is  pink. 

2.  Presence  of  oxygen  is  necessary  in  most  cases  for 
pigment  to  be  developed.  Make  two  gelatin  stab  cultures 
of  Bacillus  fluorescens  liquefaciens.  Incubate  one 
aerobically  and  the  other  anaerobically.  Examine  in  forty- 
eight  hours.  The  aerobic  culture  is  pigmented,  the  other 
is  not. 

10  3.  A  few  require  absence  of  oxygen.  Make  gelatin 
stab  of  Spirillum  rubrum  and  incubate  at  200  C.  for  3  to  4 
days,  when  growth  is  found  in  depth  to  be  red  and  on 
surface  to  be  white. 


GENERAL    PRINCIPLES  159 

Colour  Reduction  is  measured  by  the  addition  of  some 
easily  discoloured  substance  to  the  medium.  Litmus, 
methylene-blue,  and  sodium  sulphindigotate  are  used.  As 
the  bacteria  grow,  the  colour  is  discharged  in  the  anaerobic 
parts  of  the  culture.  In  a  fluid  medium,  shaking  restores 
the  colour. 

Proteinochrome  formation  is  observed  in  5  per  cent 
peptone  broth  or  3  per  cent  peptone  water.  Add  a  few 
drops  of  acetic  acid  and  then  fresh  chlorine  water,  when 
a  red-violet  colour  indicates  proteinochrome  formation. 

Test  for  Indol  Formation. — To  a  pure  culture  in 
broth  or  peptone  water  add  1  c.c.  of  o-oi  per  cent  sodium 
nitrite  solution  and  1  c.c.  of  purest  sulphuric  (25  per  cent), 
or  HC1.  A  red  coloration  within  five  minutes  indicates 
that  indol  is  present.  A  second  test  which  gives  a  positive 
result  with  B.  coli  within  forty-eight  hours  at  370  C.  is  to 
add  1  c.c.  of  an  acid  solution  of  paradimethylamido- 
benzaldehyde,  when  a  rose  or  cherry-red  colour  develops 
in  2  or  3  minutes  if  indol  is  present.  (Solution  A. :  para. 
8  grm.,  HC1  160  c.c,  absolute  alcohol  760  c.c.  ;  Solution 
B. :  cold  saturated  solution  of  potassium  sulphate.  Use 
1  c.c.  of  A,  and  shake,  and  add  1  c.c.  of  B.  and  allow  to 
stand.)  If  there  is  any  suspicion  of  the  micro-organism 
having  the  power  of  reducing  nitrates  to  nitrites,  add  the 
sulphuric  acid  first  and  wait ;  if  no  coloration  develops, 
then  add  the  nitrite  solution. 

Spirillum  cholerae  has  this  power,  and  hence  the  addition 
of  the  sulphuric  acid  is  alone  required.  This  is  called 
the  nitroso-indol  reaction. 


LIQUEFACTION     OF     GELATIN. 

This  is  due  to  the  development  of  enzymes  from  the 
growth  of  bacteria  in  proteid  media.  These  proteolytic 
enzymes  are  not  always  secretions  of  the  bacterial  cell, 
but  are  in  some  cases  closely  bound  to  the  cell-body,  and 
are  separable  from  it  only  after  its  death.  When  they  are 
true  secretory  products,  they  can  be  separated  from  the 
micro-organisms  by  filtration  through  a  Berkefeld  filter 
candle,  and  from  such  filtrates  they  can  be  obtained  in 


160         PUBLIC    HEALTH    BACTERIOLOGY 

the  dry  state  by  precipitation  with  alcohol.  Such  enzymes 
are  usually  more  thermostabile  than  when  in  solution. 
Thus,  most  enzymes  are  readily  destroyed  in  solution  at 
700  C,  but  dry  enzymes  may  withstand  1400  C.  for  10 
minutes.  (As  usual,  moist  heat  is  more  effective  than 
dry  heat.) 

This  proteolytic  (protein-splitting)  or  peptonizing  power 
varies  for  the  different  proteids,  and  is  usually  tried  on 
gelatin,  blood  fibrin,  and  casein  of  milk.  Thus,  Staphylo- 
coccus pyogenes  liquefies  gelatin  and  blood-serum,  and 
clots  milk,  but  does  not  dissolve  the  casein  ;  Streptococcus 
pyogenes  does  not  liquefy  gelatin,  nor  blood  serum,  nor 
casein  ;  B.  coli  communis  is  likewise  negative  to  all  three 
tests  ;  some  varieties  of  B.  proteus  are  positive  to  all 
three ;  B.  pyocyaneus  is  positive  to  the  three ;  Spirillum 
cholerae  liquefies  gelatin  and  blood  serum,  but  not  casein  ; 
and  so  on.  These  tests  are  still  very  useful  in  dividing 
the  bacteria  into  groups,  and  so  narrowing  the  field  in  the 
difficult  task  of  concluding,  with  moderate  certainty,  the 
race  of  a  particular  germ. 


Gelatin — liquefying 

Gelatin — non-liquefying 

Staphylococci 
B.  anithracis 

Streptococci 
Pneumococci 

B.  tetani  and  botulinus 

M.  tetragenus  and  melitensis 

B.  enteritidis  sporogenes 

B.  oedematis  maligni 

Sp.  choleras  and  most  Sp. 

Colon -typhoid  group 
B.  diphtheriae 
B.  mallei 

B.  cloacae 
B.  proteus 
B.  subtilis 

B.  pestis 

Friedlaender's  pneumobacillus 

Yeasts  (most) 

B.  pyocyaneus 
Actinomyces 
Moulds  (most) 

Note. — Organisms  which  do  not  grow  on  gelatin  or  at  air  temperature  cannot 
be  thus  classified. 

HEMOLYSIS. 

Haemolysis  will  be  treated  of  under  immunity,  but  the 
present  reference  is  to  the  haemolytic  action  of  certain 
organisms  when  grown  on  blood-agar  plates.  (Blood  agar 
is  made  from  defibrinated  blood  1  part,  and  agar  2  parts.) 


GENERAL    PRINCIPLES  161 

In  such  a  medium,  haemolysis  (destruction  of  the  red 
blood-cells)  is  shown  by  a  yellow  transparent  halo  around 
the  colonies.  Organisms  producing  hemolysins,  are : — 
Staphylococci,  Streptococci,  some  Spirilla  (but  not  Sp. 
cholera). 

STAINING     REACTIONS     AND    METHODS. 

Saturated  alcoholic  solutions  are  kept  as  stock,  and 
diluted  i  in  10  with  water  as  required,  and  filtered. 
Rather  use  dilute  stains  and  for  a  longer  time,  than  have 
precipitate  of  stain  on  preparation.  Stains  can  be  reduced 
in  intensity  if  necessary  by  using  dilute  acids,  commonly 
acetic  i  per  cent.  Acid  stains,  like  eosin,  stain  the  proto- 
plasm of  cells,  whereas  basic  stains,  like  gentian-violet, 
methylene-blue,  and  fuchsin,  stain  the  cell-nuclei  and 
bacteria. 

Blood,  pus,  and  smears  from  agar  plates  stain  most 
sharply  with  methylene-blue,  but  stain  fades  rapidly  on 
keeping.     Certain  bacteria  need  special  stains. 

Loeffler's  Methylene-blue. — Saturated  alcoholic  solution 
methylene-blue  30  c.c.  ;  solution  KOH  (o-oi  per  cent) 
100  c.c.     Keeps  well. 

Aniline  Oil-Water  Stains. — Made  with  saturated  alco- 
holic solutions  of  gentian-violet  and  fuchsin,  which  are 
mixed  1  in  10  of  aniline  oil-water.  The  latter  is  a  mixture, 
made  by  shaking  5  c.c.  of  aniline  oil  in  100  c.c.  distilled 
water ;    filter,  and  keep  in  dark.     These  keep  badly. 

Carbol- fuchsin  (Ziehl-Neelsen) . — Ac.  carbolic  (5  per 
cent)  100  c.c.  ;  saturated  alcoholic  solution  fuchsin  10  c.c. 
Diluted  3  to  4  times  it  stains  more  slowly  but  better. 
Keeps  well. 

Carbol-glycerin- fuchsin. — Fuchsin  1  grm.,  ac.  carbolic 
liq.  5  c.c,  glycerin  50  c.c,  and  aq.  dest.  100  c.c  Dilute 
in  use  4  to  10  times.     Keeps  well. 

Carbol-methylene-blue. — Methylene-blue  1-5  grm.,  abso- 
lute alcohol  10  c.c,  ac  carbolic  (5  per  cent)  100  c.c. 
Keeps  well. 

To  Make  a  Film. — Take  a  cover-slip  (in  Cornet's  for- 
ceps), and  put  on  it  a  drop  of  distilled  water  (small  for  fear 
of  plasmolysis) .  Take  two  strokes  of  culture  with  platinum 
needle  and  rub  into  drop,   and  spread  out.     Dry  in  air 

11 


162 


PUBLIC    HEALTH    BACTERIOLOGY 


(should  dry  at  once).  Fix  by  passing  three  times  through 
the  flame.  Stain  for  two  or  three  minutes.  Wash  with 
water  and  examine  on  clean  slide  in  water-drop  (using  oil 
immersion).  In  water-drop  bacteria  look  larger,  can  be 
restained,  and  can  be  kept  longer  than  when  mounted 
direct  in  Canada  balsam.  To  preserve  :  allow  to  dry, 
remove  from  slide,  roll  up,  and  label. 

Counterstain  with  eosin  or  other  stain  in  dilute  solution 
for  i  to  2  minutes.     Eosin  stains  cell  protoplasm  red. 

Films  are  also  made  on  slides,  which  are  more  easily 
handled  than  coverslips. 

Gram's  Method  of  Staining — Depends  on  the  fact 
that  some  bacteria  when  well  stained  retain  the  stain  after 
treatment  with  a  solution  of  iodine  and  subsequent  washing 
with  alcohol  (strong  or  absolute).  This  is  believed  to  be 
due  to  the  stain  and  the  iodine  forming  a  combination 
which  resists  decoloration. 

Table. 


Gram-positive 

Gram-negative 

(Retain  the  gentian-violet) 
Staphylococcus  pyogenes 
Streptococcus            ,, 
Pneumococcus 

(Take  the  counterstain) 
Meningococcus 
Gonococcus 
Micrococcus  melitensis 

Micrococcus  tetragenus 
Bacillus  anthracis 
subtilis 
diphtheriae 
tetani 

catarrhalis 
Colon-typhoid  group  of  bacilli 
Cholera  group  of  spirilla 
Bacillus  pestis  and  group 
,,       mallei 

botulinus 

influenzas 

tuberculosis  and 
other  acid-fasts 

aerogenes     capsu- 
latus 

pyocyaneus 
,,        proteus 
„        Koch- Weeks 

Morax  -  Axenf  eld 

„       enteritidis    sporo- 
genes 
of  swine  erysipelas 

maligni  cedematis 
anthracis  symptomatici 
of  fowl  cholera 

of  mouse  septi- 
,,           caemia 
,,       of  potato 
Yeasts  and  many  moulds 

of  rabbit  septicaemia 
Spirillum  Obermeieri  (spirochaete) 
Friedlaender's  diplobacillus 

Streptothrix  actinomyces 

Method. — Stain  for  5  minutes  with  aniline-oil-gentian- 
violet,  or  carbol-gentian-violet.     Pour  off  excess  of  stain 


GENERAL    PRINCIPLES  163 

and  cover  with  Lugol's  (or  Gram's)  solution  of  iodine 
(iodine  I,  KI  2,  aq.  dest.  300)  for  30  seconds  to  2  minutes. 
Wash  with  97  per  cent  alcohol  (or  methylated  spirit)  until 
washings  are  no  longer  coloured  (takes  about  30  sec.  to 
2  min.).  Examine  in  water,  or  dry  and  mount  in  balsam. 
To  counterstain :  remove  alcohol  with  water  and  cover 
with  dilute  carbol-fuchsin  for  a  few  seconds  (or  saturated 
watery  solution  of  Bismarck  brown  for  longer).  Wash  in 
water,  dry,  and  mount.  Result :  Bacteria  blue-black,  or 
colourless,  or  red  ;  tissues  red.  Those  bacteria  which  are 
blue-black  are  said  to  be  Gram-staining  or  Gram- positive. 
The  others  are  said  to  be  Gram-negative.  (See  Table  on 
previous  page.) 

Acid-fast  Bacteria. — Some  bacilli  stain  with  difficulty 
with  ordinary  dyes,  requiring  the  aid  of  heat  or  a  mordant 
(as  carbolic).  Such  bacilli  usually  retain  the  stain  even 
when  treated  with  dilute  acids  and  alcohol,  and  hence  are 
called  "  acid-proof  "  or  "  acid-fast."  This  resistance  is 
believed  to  be  due  to  the  presence  in  the  cell-body  of  a 
waxy  substance  (an  alcohol).  The  members  of  this  group 
are :  Bacilli  of  human,  bovine,  avian,  and  fish  tubercu- 
losis; Moeller's  Timothy-grass  bacilli  (1)  and  (2)  ;  Mist- 
bacillus;  Rabinowitch's  butter  bacillus;  Korn's  butter 
bacilli  (2)  and  others ;  Johne's  bacillus  (of  chronic  bovine 
pseudo-tuberculous  enteritis) ;  Bacillus  smegmatis  (smegma 
bacillus);  Bacillus  leprae  (leprosy  bacillus). 

Method. — Flood  slide  or  cover-glass  with  carbol-fuchsin 
and  heat  for  3  minutes.  Wash  and  decolorize  by  dipping 
into  5  per  cent  sulphuric  acid  and  60  per  cent  alcohol  alter- 
nately until  film  looks  colourless.  Wash  in  water.  Counter- 
stain  with  aqueous  methylene-blue  for  a  half  to  one  minute. 
Wash  and  examine.  The  acid-fast  bacteria  are  stained  red, 
while  the  others  and  the  matrix  are  stained  blue. 

Alcohol-fast  Bacteria. — In  specimens  of  urine  being 
examined  for  tubercle  bacilli,  acid-fast  smegma  bacilli  may 
also  be  present.  To  distinguish:  counterstain  film  in  a 
saturated  solution  of  methylene-blue  in  absolute  alcohol 
for  5  minutes.  Tubercle  bacilli  remain  red,  while  smegma 
bacilli  become  blue. 

Capsule  Staining. —  Many  bacteria  possess  a  mucoid 
or  gelatinous  envelope,  though  it  is  only  in  a  few  species 


164         PUBLIC    HEALTH    BACTERIOLOGY 

that  it  is  easily  demonstrable.  It  is  known  as  the 
"  capsule  "  and  varies  in  thickness  from  being  only  just 
visible  to  4  or  5  times  the  size  of  the  bacterium  itself.  It 
is  mostly  seen  in  preparations  taken  directly  from  animal 
tissues  or  fluids  or  exudates,  or  from  cultures  in  media 
containing  animal  serum  or  milk.  It  is  best  seen  in  the 
Diplococcus  pneumoniae,  Micrococcus  tetragenus,  B. 
aerogenes  capsulatus,  and  the  bacilli  of  the  Friedlaender 
group.  Hiss's  method :  Make  a  cover-slip  film,  and 
preferably  by  using  a  drop  of  animal  serum  instead  of 
water.  Dry  in  air  and  fix  by  heat.  Stain  for  a  few 
seconds  with  dilute  fuchsin  or  gentian-violet  (1  of  saturated 
alcoholic  solution  in  19  of  aq.  dest.),  meanwhile  heating 
the  preparation  over  a  flame  until  steam  arises.  Wash 
off  dye  with  20  per  cent  watery  solution  of  copper 
sulphate.  Blot  dry  (do  not  wash  with  water),  and  mount 
direct  in  Canada  balsam.  The  capsule  appears  as  a  faint 
blue  halo  around  a  dark  purple  cell-body. 

Spore  Staining. — Prepare  film  as  usual  and  fix  in  the 
flame.  Place  in  CHC13  for  two  minutes.  Wash  in  water. 
Place  in  5  per  cent  chromic  acid  for  half  a  minute  to  two 
minutes.  Wash  in  water.  Float  cover-slip,  film  side  down, 
on  carbol-fuchsin  solution  in  a  small  porcelain  basin  and 
heat  stain  gently  until  it  steams  ;  continue  in  stain  for  3  to 
5  minutes.  Decolorize  in  5  per  cent  sulphuric  acid  for  5  to 
10  seconds.  Wash  in  water.  Stain  with  saturated  watery 
methylene-blue  for  30  to  60  seconds.  Wash  and  examine ; 
or  dry,  and  mount  in  balsam.  The  spores  are  stained  red 
and  the  cell  bodies  blue. 

Spores  are  believed  to  be  an  encysted  or  resting  stage 
of  bacteria,  and  not  a  method  of  reproduction,  or  rather 
multiplication.  In  most  cases  only  one  spore  is  produced 
by  one  bacillus,  and  the  latter  becomes  extinct  when  the 
spore  is  fully  developed.  Spore  formation  is  not  very 
common  among  bacteria,  and  is  found  almost  exclusively 
among  the  bacilli,  less  commonly  in  the  spirilla,  and  rarely, 
if  at  all,  in  micrococci.  The  anaerobic  bacilli  are  almost 
all  spore-forming,  but  amongst  aerobes  the  only  sporing 
bacterium  pathogenic  to  man  is  the  anthrax  bacillus. 
This  materially  facilitates  and  simplifies  the  disinfection 
and  treatment  of  infectious  diseases,  as  spores  are  extremely 


GENERAL    PRINCIPLES  165 

resistant  to  injury  by  heat,  light,  drying,  and  chemicals. 
True  spores  or  endospores  are  to  be  distinguished  from 
arthrospores,  the  existence  of  which  is  now  seriously 
questioned.  An  arthrospore  is  a  bacterium  which  enters 
into  a  resting  stage  without  any  new  formation  within 
the  protoplasm.  It  stains  well  with  ordinary  stains,  and 
has  no  distinct  capsule,  but  is  stated  to  have  increased 
resistance  to  external  agents.  A  true  spore  (i)  resists  the 
ordinary  staining  method ;  and  (2)  shows  very  great 
resistance  to  destruction  to  the  usual  agents. 

Flagella  Staining.  —  Flagella  are  hair-like  organs 
used  for  locomotion,  and  have  been  described  as  occurring 
on  bacilli,  spirilla,  and  a  few  species  of  cocci.  They  are 
best  seen  in  young  cultures,  10  to  18  hours  old,  at  370  C. 
McCrorie's  method  gives  admirable  results  when  the 
technique  is  carefully  followed. 

McCrorie's  Flagella  Stain. — Measure  out  and  mix  : — 
Night-blue,  1  grm.  in  20  c.c.  of  absolute  alcohol ;  potash 
alum,  1  grm.  in  20  c.c.  of  distilled  water  ;  tannin,  1  grm. 
in  20  c.c.  of  distilled  water.  Allow  mixture  to  stand  for 
twenty-four  hours,  and  filter  supernatant  fluid.  Keep 
stain  in  incubator  and  filter  again  when  using. 

Method. — 

(1)  Take  some  distilled  water  at  370  C.  in  a  watch- 

glass  ;  place  therein  a  loopful  of  young  agar  cul- 
ture, and  allow  to  swim  off  and  diffuse  without 
stirring. 

(2)  Take  several  loopfuls  of  this  solution,  and  deposit 

them  singly,  without  smearing,  on  a  clean  cover- 
slip. 

(3)  Dry  in  the  incubator  at  370  C. 

(4)  Apply  stain  (also  at  370  C.)  and  replace  in  incubator 

for  10  minutes. 

(5)  Wash  off  stain  by  dipping   cover-slip   edgeways 

several  times  into  water  at  370  C. 

(6)  Dry  in  the  incubator. 

(7)  Mount :  or  counterstain  bodies  with  strong  fuchsin 

solution    for    2    minutes ;   wash,    and    dry    as 
before  ;   mount. 
Flagella  are  blue,  bacillary  bodies  are  red. 


166         PUBLIC    HEALTH    BACTERIOLOGY 

Granules. — Diphtheria  bacilli  when  stained  show  oval 
bodies,  which  stain  more  deeply  than  the  rest  of  the  cell. 
Loeffler's  methylene-blue  (page  161)  shows  them  well,  but 
a  contrast  stain  is  often  used,  such  as  that  of  Neisser. 

Neisser's  Method. — Two  solutions  are  used  :  Solution  i : 
methylene-blue  I  grm.  +  20  c.c.  alcohol  (96  per  cent)  + 
50  c.c.  glacial  acetic  acid  +  950  c.c.  aq.  Solution  2 : 
Bismarck-brown  2  grm.  dissolved  in  1  litre  of  boiling 
distilled  water.  Make  a  film,  fix,  stain  with  Solution  1 
for  30  to  60  seconds.  Wash,  and  pour  on  Solution  2,  and 
after  30  seconds  wash  off  with  water.  Dry,  and  mount. 
Bodies  of  the  bacilli  are  brown,  and  the  granules  are  blue. 

Paraffin- section  Staining.  —  Sections  must  first  be 
fixed  on  slides  by  one  of  two  modes  : — 

1.  Float  section  on  warm  water  (under  400  C.J,  insert 
slide  underneath,  with  a  needle  fix  one  corner,  and  withdraw 
slide.     Dry  for  24  hours  in  incubator  at  370  C. 

2.  Place  a  drop  of  solution  of  egg-white  (10  per  cent  in 
aq.)  on  a  slide,  draw  on  section  as  before,  and  incubate  at 
370  C.  for  30  minutes ;  or,  remove  excess  of  moisture,  heat 
over  small  flame  until  paraffin  melts,  and  then  until 
vapour  arises. 

Staining.     General  Method. — 

1.  Preparation  :     Remove   paraffin   with   xylol,    and 

xylol  with  absolute  alcohol.  Wash  in  water 
(unless  alcoholic  solution  of  stain  is  used). 

2.  Staining :    Use   methylene-blue    for    15   minutes ; 

carbol-thionin-blue  (5  minutes),  or  aniline-oil- 
gentian  violet,  carbol-fuchsin,  etc.  For  over- 
staining  reduce  with  very  weak  acid  for  5  to  30 
seconds.     This  also  decolorizes  the  tissues. 

3.  Counter-staining :    WTash  in   water,    stain   with    \ 

per  cent  eosin  for  30  seconds,  and  wash  in  water. 

4.  Dehydration  and  Clearing :    Remove  water  with 

absolute  alcohol  (some  organisms  are  decolorized 
very  easily  at  this  stage,  and  hence  treatment 
must  be  rapid).  Remove  alcohol  with  xylol, 
and  mount  in  Canada  balsam. 
Weigert  advises  aniline  oil,  aniline-xylol,  xylol,  and 
balsam. 


GENERAL    PRINCIPLES  167 

Gram's  Method. — (i)  Prepare  ;  (2)  Stain  for  5  minutes 
with  aniline-oil-gentian-violet ;  (3)  Pour  off  excess,  do  not 
wash,  flood  with  Gram's  iodine  solution  repeatedly,  until 
purplish  black,  and  allow  to  act  for  1  minute  ;  (4)  Do  not 
wash,  but  decolorize  with  absolute  alcohol  or  methylated 
spirit  until  faint  violet  tint ;  (5)  Wash  in  water,  counter- 
stain  with  J  per  cent  eosin  for  1  minute  ;  (6)  Dehydrate, 
clear,  and  mount.  Bacteria  are  blue-black,  and  tissues 
are  pink. 

In  Weigert's  modification  of  the  Gram  method,  the 
section  is  first  stained  for  30  minutes  in  lithia-carmine. 
Wash  in  water  and  proceed  as  in  Gram's  method,  except 
that  the  dehydration  is  done  with  aniline  oil. 

Acid- fast  Bacilli  in  sections. — Tubercle  bacilli,  etc. 

(1)  Prepare  ;  (2)  Stain  in  carbol-fuchsin  for  5  minutes  in 
hot.  solution,  or  24  hours  in  cold ;  (3)  Wash  in  water  ; 
(4)  Decolorize  in  12  per  cent  sulphuric  acid  ;  (5)  Wash 
well  in  water  (colour  should  just  be  a  faint  pink)  ;  (6) 
Contrast  stain  with  saturated  watery  solution  of  methylene- 
blue  for  30  seconds ;  (7)  Wash  in  water,  dehydrate,  clear, 
and  mount.     Bacilli  are  red,  tissues  are  blue. 

Note. — If  a  section  has  been  hardened  in  corrosive 
sublimate,  the  latter  must  be  removed  after  the  paraffin. 
This  is  done  by  using  equal  parts  of  Gram's  solution  and 
water  for  a  few  minutes,  and  then  removing  the  iodine 
with  methylated  spirit. 

POLYCHROME     STAINS. 

These  are  of  value  for  the  staining  of  micro-organisms  in 
pus  and  exudates,  and  for  blood  films,  in  all  of  which  the 
relation  of  the  bacteria  or  protozoa  to  the  cellular  elements 
is  to  be  determined.  In  all  these  stains  the  basis  is  a 
mixture  of  solutions  of  methylene-blue  and  eosin,  which 
stain  the  various  elements  separately  and  in  combination, 
thus  bringing  out  in  a  marvellous  way  the  details  of  the 
structural  and  foreign  bodies.  This  mixture  is  called  the 
Romanowsky  stain,  and  various  modifications  of  it  are 
now  in  use. 

Jenner's  Stain. — A  simple  stain,  excellent  for  blood  work, 
but  not  so  good  for  parasites  as  others  given  below.     No 


168         PUBLIC    HEALTH    BACTERIOLOGY 

alkali  is  used  in  its  preparation.  Equal  parts  of  watery 
solutions  of  (a)  Gruebler's  water-soluble  eosin  (i-2  per  cent), 
and  (b)  Gruebler's  medicinal  methylene-blue  (i  per  cent), 
are  mixed,  and  the  mixture  allowed  to  stand  for  twenty- 
four  hours.  A  coarse  granular  precipitate  forms,  is  filtered 
off,  dried  at  550  C.,  washed  with  distilled  water,  filtered, 
washed  again,  filtered,  and  dried.  Of  the  dried  powder, 
0-5  grm.  is  dissolved  in  100  c.c.  of  Merck's  methyl  alcohol. 
In  use,  a  few  drops  are  placed  on  the  dried  unfixed  film  for 
one  to  three  minutes,  then  poured  off,  and  the  slide  is 
washed  with  distilled  water  until  pink  in  colour.  Dry  with 
filter-paper,  and  mount  in  xylol  balsam. 

Leishman's  Stain. — The  methylene-blue  is  alkalinized 
with  0-5  per  cent  of  sodium  carbonate,  weaker  eosin  oolution 
is  used,  and  the  technique  of  preparation  is  varied.  The 
stain  will  keep  for  a  long  period.  In  use,  a  few  drops  are 
placed  on  the  unfixed  preparation  for  fifteen  to  thirty 
seconds,  the  film  being  tilted  from  side  to  side  to  prevent 
drying  at  any  part.  In  this  way  the  film  is  both  fixed  (by 
the  methyl  alcohol)  and  stained  by  one  operation.  About 
twice  as  much  distilled  water  is  now  added,  and  the  diluted 
stain  allowed  to  act  for  five  minutes  longer.  Now  wash 
in  distilled  water,  mount,  and  examine. 

Giemsa's  Stain  and  Method. — This  is  a  modified 
Romanowsky,  of  great  value  in  staining  Spirochseta  pallida, 
Vincent's  spirilla,  protozoa,  and  Negri  bodies.  The  stain 
used  is  methyl-azure,  which  Giemsa  believes  to  be  the  essen- 
tial constituent  of  the  Romanowsky  stain.  In  use,  the  film 
is  first  fixed  with  alcohol,  dried,  covered  with  the  stain,  dilu- 
ted, well  washed,  drained,  dried,  and  mounted.  For  ordinary 
staining,  fifteen  minutes  are  enough ;  for  the  spirochete  and 
Negri  bodies,  one  to  twelve  hours  may  be  necessary. 

Staining. —  By  Romanowsky,  red  cells  are  stained 
orange  to  pink  ;  eosinophile  granules,  red  ;  neutrophile 
granules,  yellow  to  lilac  ;  nuclei,  shades  of  violet  ;  blood 
platelets,  purplish  ;  malarial  parasites,  blue  ;  chromatin, 
red  to  rose-pink. 

Blood  Films. — May  be  made  on  cover-slips  or  on  slides. 
With  cover-slips,  touch  one  to  the  exuding  blood,  drop 
it  on  another,  and  then  draw  the  cover-slips  apart.  For 
slides,  touch  a  drop  of  blood  near  one  end  of  slide,  and  smear 


GENERAL    PRINCIPLES  169 

out  with  another  by  drawing  it  slowly  along  the  first,  sloped 
to  it  at  an  angle  of  about  450.  The  slides  used  must  be 
clean,  and  are  usually  stored  in  absolute  alcohol,  which 
is  burnt  off  just  when  using.  When  the  film  is  stained, 
it  can  be  examined  at  once,  with  a  drop  of  cedar  oil,  and 
afterwards  mounted,  if  desired. 

Fixation  is  accomplished  by  methyl  alcohol  after  air- 
drying,  when  using  the  Romanowsky  method.  For  other 
processes  it  is  attained  by  one  of  the  following  methods  : — 

(a).  Half  an  hour  in  a  hot-air  chamber  at  1200  C. 

(b).  Half  an  hour  in  a  mixture  of  alcohol  and  ether 
(equal  parts).     Wash,  and  dry. 

(c).  Five  minutes  in  formol-alcohol  (1—9).  Wash,  and 
dry  (Gulland). 

(d).  Two  to  three  minutes  in  saturated  solution  of 
corrosive  sublimate.     Wash  well,  and  dry. 

For  wet  films,  which  give  the  histology  better,  the  methods 
are  varied.  The  films,  while  still  wet,  are  placed  film 
downwards  in  the  fixative.  Gulland's  combination  of  (b) 
and  (d)  is  said  to  be  an  excellent  one. 

INOCULATION     OF     ANIMALS. 

Inoculation  of  animals,  or  the  animal  experiment,  as  it 
is  called,  is  used  for  a  variety  of  purposes,  is  made  in  a 
variety  of  ways,  and  the  number  of  different  animals  used 
is  now  considerable.  It  is  an  important  way  of  getting  a 
pure  culture  in  difficult  cases.  It  also  determines 
the  pathogenicity  of  a  pure  culture  injected.  By  the 
occurrence  of  special  symptoms  following  injection  of 
suspected  material,  it  serves  to  establish  the  presence  of  a 
particular  micro-organism  in  the  material.  If  the  injection 
of  known  products  of  certain  organisms  is  followed  by 
certain  reactions,  the  presence  in  the  animal's  body  of 
the  organism  from  which  the  product  has  been  derived  is 
inferred  (tuberculin  and  mallein  tests) .  By  passing  a  par- 
ticular organism  through  a  series  of  susceptible  animals 
in  succession,  its  virulence  may  be  exalted,  and  the  same 
experiment  through  resistant  animals  may  depress  its 
vitality  ;  this  is  Pasteur's  "  method  of  passage."  Animal 
inoculation  is  also  used  for  the  production  of  anti- 
toxins and  antibacterial  bodies. 


170 


PUBLIC    HEALTH    BACTERIOLOGY 


The  inoculation  may  be  :  (i)  Cutaneous,  that  is,  rubbing 
into  the  unbroken  skin  ;  (2)  Subcutaneous,  with  a  syringe, 
or  by  cutting  the  skin  and  putting  the  material  in  a  pocket 
in  the  subcutaneous  tissue  and  stitching  up  the  skin  wound  ; 
(3)  Intraperitoneal ;  (4)  Intramuscular ;  (5)  Intrapleural ; 
(6)  Intravenous ;  (7)  Into  the  stomach  by  a  tube,  or  by 
ordinary  feeding  ;    and  (8)  By  inhalation. 

The  various  processes  are  described  as  required  under 
the  particular  microbes  concerned.  The  average  tempera- 
tures of  the  more  commonly  used  animals  and  a  few 
others  may  be  here  conveniently  tabulated.  The  table 
is  compiled  from  Abel's  "  Laboratory  Handbook  of 
Bacteriology,"  and  from  various  other  sources. 


Table  of  Animal  Temperatures. 


Rectal  Temperature 

Pulse 

Centigrade 

Fahrenheit 

Rate 

Where  usually 
observed 

Guinea-pig 

37-3°  to  39-5° 

99°  to  1030 

— 



Horse 

37'7°  to  38-3° 

ioo°  to  IOI° 

35  to  45 

jaw 

Cow 

377°  to  38  90 

IOO°  to  102° 

45  to  55 

jaw 

Calf 

38-4°  to  39-9° 

1010  to  103-8° 

— 

— 

Sheep 

38-8°  to  40° 

102°  tO   IO40 

70  to  80 

heart 

Pig 

ditto 

ditto 

ditto 

ditto 

Goat 

38-6°  to  3970 

ioi#4°to  103-4° 

— 

— 

Dog 

38-6°  to  39'5° 

101-4°  to  103° 

80  to  90 

— 

Cat 

377°  to  38-3° 

ioo°  to  IOI° 

— 

— 

Rabbit 

38-3°  to  39-9° 

ioi°  to  104° 

— 

— 

Chicken 

410  to  42-5° 

105°  to  108-5° 

— 

— 

Pigeon 

ditto 

ditto 

- 

— 

Linnet 

44° 

111° 

— 

— 

Rhesus 

Monkey. . 

38-1°  to  39-5° 

100-5°  to  103° 

— 

— 

Chimpanzee 

37°  to  380 

98-4°  to  100-4° 

— 

— 

Bat 

4i 

106° 

— 

— 

Narwhal    .  . 

35-5° 

96° 

— 

— 

Reptiles     . . 

28° 

82-5° 

— 

— 

UNICELLULAR    MICRO-ORGANISMS. 

Fungi. — Fungi  are  members  of  the  class  of  plants  called 
Thallophyta,  which  show  no  division  into  root  and  stem. 
They  are  distinguished  from  the  algae  of  the  same  class  by 
not  possessing  chlorophyll. 


GENERAL    PRINCIPLES  171 

Schizoniycetes,  or  fission-fungi — multiply  by  fission : 
coccus,  bacillus,  spirillum,  streptothrix. 

Blastomycetes,  or  budding-fungi — multiply  by  budding: 
the  yeasts  or  torulae. 

Hyphomycetes ,  or  branching-fungi — multiply  by  branch- 
ing. The  branches  are  called  hyphae,  and  the  network  of 
interlacing  threads  is  called  the  mycelium  :  the  moulds. 

Protozoa. — Unicellular  members  of  the  animal  kingdom. 
They  are  divided  into  groups  like  the  following  : — 

Sarcodina. — Naked  or  cased  organisms  which  throw  out 
pseudopodia :  amceba. 

Flagellata. — Endowed  with  organs  of  locomotion — 
flagella :  trypanosoma,  spirochaeta. 

Infusoria. — Locomotion  by  means  of  short  flagella, 
called  cilia,  of  which  many  are  present :  balantidium. 

Sporozoa. — Non-motile  in  adult  state.  Reproduce  by 
spores.  Feed  by  osmosis.  Exclusively  endoparasites : 
Plasmodium  malariae  or  haemosporidia,  piroplasma, 
coccidium. 

Schizomycetes  may  be  classed  in  various  ways  under 
the  following  heads  : — 

Parasites  or  Saprophytes 

Aerobes  or  Anaerobes 

The  parasites,  saprophytes,  aerobes,  and  anaerobes  may 
be  either  obligatory  or  facultative. 

Pathogenic  or  Non-pathogenic 

Sporing  or  Non-sporing 

Motile  or  Non-motile 

Flagellated  or  Non-flagellated 

Gelatin-liquefying     or  Non-liquefying 

Gram-staining  or  Non-Gram-staining 

Chromogenic  or  Non-chromogenic. 

Other  characters  used  to  distinguish  bacteria  into  groups 
are :  their  action  on  the  various  sugars,  the  production  of 
indol  in  certain  media,  reduction  of  nitrates,  their  behaviour 
to  the  dyes,  e.g.,  acid-fast  or  not,  polar  staining,  etc. 


CHAPTER  X. 
RESULTS     OF     BACTERIAL     ACTIVITY. 

PRODUCTS. 

These  result  mainly  from  the  cleavage  of  proteids  and 
fats,  and  the  fermentation  of  carbohydrates.  The  basis 
of  our  knowledge  on  this  subject  was  laid  by  Pasteur,  who 
also  was  the  first  to  prove  the  part  played  by  micro- 
organisms in  these  processes.  The  actual  work. of  cleavage 
is  carried  out  by  ferments  or  enzymes.  A  ferment  or 
enzyme  is  a  substance  produced  by  a  living  cell,  which 
substance  is  able  to  bring  about  enormous  chemical  change 
(in  proportion  to  its  bulk)  without  itself  suffering 
decomposition.  The  accumulation  of  its  products  often 
causes  its  action  to  cease,  but  if  these  are  removed,  the 
action  is  indefinitely  prolonged.  We  shall  see  that  the 
toxins  of  bacteria  have  been  compared  to  enzymes,  and 
while  to  some  extent  there  is  a  resemblance  in  their  action, 
the  toxins  in  a  certain  amount  are  able  to  produce  only  a 
definite  result,  which  is  less  than  that  produced  by  a 
larger  dose. 

The  various  enzymes  are  grouped  as  proteolytic  (in 
culture,  gelatin-,  fibrin-,  serum-liquefying),  fat-splitting, 
and  carbohydrate  -  splitting  (produce  alcohol,  simpler 
sugars,  lactic  acid,  butyric  acid,  acetic  acid). 

Other  activities  are  :  denitrification,  nitrification,  light- 
production,  colour-production,  sulphur-utilization  (sulphur 
bacteria),  etc. 

Ptomaines. — The  action  of  bacteria  on  dead  animal  mat- 
ter by  their  proteolytic  enzymes,  produces  substances  called 
ptomaines,  or  "  animal  alkaloids."  These  bodies  are  toxic 
to  the  human  species  (and  others) ,  and  are  organic  chemical 
compounds,  basic  in  nature,  which  combine  with  acids  to 
form  salts.  They  have  to  be  distinguished  from  leuco- 
maines,  similar  substances  formed  in  the  living  body  during 
proteid  metabolism,  and  not  by  bacterial  action.  They 
have  also  to  be  distinguished  from  the  bacterial  toxins, 


BACTERIAL    ACTIVITY  173 

which  are  developed  by  bacterial  growth,  independent  of 
the  medium  in  which  grown,  and  have  even  been  obtained 
in  cultures  in  proteid-free  media.  Of  the  ptomaines, 
putrescin  and  cadaverin  are  extremely  poisonous,  and  most 
cases  of  meat-poisoning,  cheese-poisoning,  and  vegetable- 
poisoning  are  due  to  one  or  another  of  these  ptomaines. 


INFECTION. 

The  invasion  of  the  animal  body  by  bacteria  is  spoken 
of  as  infection  if  it  gives  rise  to  disease.  The  definition 
requires  extension  to  cover  the  case  of  diphtheria,  where 
the  invasion  by  the  micro-organism  is  often  very  slight, 
but  where  the  disease  is  due  to  the  invasion  of  the  body  by 
the  toxins  or  bacterial  products.  In  most  cases  the 
infection  is  due  to  both  the  bacteria  and  their  products, 
in  varying  degrees. 

In  the  first  place  it  is  useful  to  note  that  the  skin  and 
the  mucous  membranes  of  the  alimentary  tract,  the  mouth, 
the  nasal  passages,  the  upper  respiratory  tract,  the  con- 
junctivae, and  the  genital  passages,  are  normally  inhabited 
by  various  species  of  bacteria.  Some  of  these  are 
facultative  parasites,  and  seize  the  opportunity  of  a  break 
in  the  surface,  or  other  injury,  to  grow  and  multiply,  and 
so  produce  disease.  Others  are  pure  saprophytes,  non- 
pathogenic in  any  circumstances  to  the  body  on  which 
they  harbour. 

The  definition  of  the  infective  diseases  will  be  useful  here. 

An  infective  disease,  or  rather  a  specific  infective  disease, 
is  one  which  results  from  the  introduction  into  the  body, 
(i)  by  wounds,  (2)  by  the  air-passages,  or  (3)  by  the 
alimentary  tract,  of  a  definite  ferment,  or  poison,  or 
micro-organism,  which  grows  and  multiplies  in  the  body. 

In  some  of  these  diseases  the  poison  is  given  off  again, 
and  they  are  then  spoken  of  as  infectious,  or  transmissible 
from  person  to  person.  Where  contact  is  necessary  for 
transmission,  they  are  called  contagious.  The  tendency 
is  to  give  up  the  use  of  these  terms,  infectious  and  con- 
tagious, and  simply  to  speak  of  infective  diseases,  which 
are  transmissible  in  various  ways,  as  by  the  air,  by  food, 
by  contact,  by  fomites,  by  insects. 


174         PUBLIC    HEALTH    BACTERIOLOGY 

They  are  called  specific,  because  they  have  a  perfectly 
definite  course,  characterized  by  the  stages  of  incubation, 
invasion,  advance  and  death,  or  decline  and  convalescence. 
Some  of  them  have  also  a  skin  eruption  or  rash. 

The  infection  is  given  off  again  by  the  breath,  exhalations 
from  the  skin  and  wounds,  by  desquamated  portions  of 
the  epidermis,  by  the  secretions  and  excretions  (mucus  of 
mouth  and  throat,  saliva,  sputa,  faeces,  urine,  seminal 
fluid,  milk). 

Micro-organisms,  then,  are  only  relatively  pathogenic  or 
non-pathogenic,  and  in  any  particular  instance  of 
pathogenicity,  the  amount  and  kind  of  attack  vary  with 
a  large  number  of  factors.  This  is  not  a  matter  for  wonder 
now,  with  our  knowledge  of  the  varied  needs  and  differences 
in  vitality  of  many  micro-organisms,  but  was  a  stumbling- 
block  in  the  early  days.  Following  Muir  and  Ritchie,  we 
may  summarize  the  matter  thus  : — 

Infection  is  conditioned  by  (i)  the  infecting  agent,  and 
(2)  the  subject. 

1.  The  infecting  agent  produces  its  effect  dependent 

on  (a)  its  virulence,  (b)  its  numbers,  (c)  its  path 
of  entrance. 

2.  The  subject  varies  in  its  susceptibility  or  the 

reverse  (resistance),  according  to  (a)  its  species, 
(b)  race,  (c)  age,  (d)  individual  peculiarities, 
(e)  vitality,  (/)  other  disease. 

Mode  of  Action. — Multiplication ;  invasion  of  lymphatics  ; 
invasion  of  blood-stream  ;  settlement  in  certain  tissues  ; 
chemical  products  (toxins),  locally  or  diffused. 

Effects  of  Bacterial  Action. 

1.  Tissue  changes  : — 

(a)  Local  —  tissue  reactions  or   degeneration    and 

necrosis,  acute  or  chronic. 

(b)  Distant — damage  to  special  tissues,  reaction  of 

blood-forming  organs. 
(c).  General — malnutrition  or  increased  waste,   or 
both. 

2.  Metabolic  changes  :   fever,  etc. 


BACTERIAL    ACTIVITY  175 

BACTERIAL     POISONS. 

The  knowledge  of  these  is  by  no  means  complete,  so 
that  sharp  distinctions  between  various  kinds  cannot  be, 
at  present,  depended  on.  The  first  to  study  their  pro- 
duction was  Brieger,  and  it  was  while  so  engaged  that  he 
was  led  to  the  discovery  of  the  ptomaine  poisons.  These 
bodies,  however,  did  not,  on  injection,  reproduce  the 
symptoms  of  diseases  associated  with  the  bacteria  con- 
cerned in  their  production,  and  so  the  ptomaines  are  not 
nowadays  classed  as  true  bacterial  poisons.  Roux  and 
Yersin,  in  1889,  filtered  broth- cultures  of  B.  diphtherias 
through  unglazed  porcelain  (Chamberland  filter),  and 
showed  that  the  filtrate  was  bacteria  free,  and  yet  on 
injection  the  filtrate  produced  practically  the  same  effects 
as  the  injection  of  the  living  bacilli.  From  this  it  was 
inferred  that  the  filtrate  contained  the  toxin  of  the 
diphtheria  bacillus.  The  same  method  applied  to  other 
bacteria  yielded  no  such  result  in  most,  and  so  the  con- 
ception was  reached  that  some  bacteria  secrete  or  excrete 
poisons  which  are  soluble  in  the  media  in  which  they  are 
grown,  and  some  do  not.  Of  the  former  class,  diphtheria 
and  tetanus  are  the  types  ;  of  the  latter,  the  tubercle 
bacillus  may  be  taken  as  a  type,  but  the  class  is  a  very 
large  one,  including  all  the  bacteria  except  diphtheria, 
tetanus,  botulinus,  and  the  anaerobes  generally  (some  to 
only  a  small  extent).  Other  bacteria,  such  as  dysentery 
and  cholera,  are  said  to  produce  soluble  poisons,  but  the 
results  are  still  discordant.  As  a  consequence  of  these 
findings,  and  of  the  further  observation  that,  in  the 
bacteria  not  secreting  soluble  poisons,  the  injection  of 
dead  bacteria  could  reproduce  many  of  the  characteristic 
lesions  of  the  disease  associated  with  them  in  the  living 
state,  the  division  of  bacterial  poisons  into  two  groups 
has  arisen,  viz  : — 

1.  Extracellular  toxins,  true  toxins,  or  soluble  toxins. 

2.  Intracellular  toxins,  endotoxins. 

After  the  removal  of  these  bodies  from  the  bacteria,  a 
certain  proteid  residue  remains,  which,  on  injection,  gives 
rise  to  localized  reactions.  Buchner  calls  this  residue 
"  bacterial  protein,"  and  believes  it  to  be  the  same  in  all 


176         PUBLIC    HEALTH    BACTERIOLOGY 

bacteria,  to  be  without  specific  toxic  action,  but  having  a 
positive  chemiotactic  action  on  the  white  cells  of  the  blood, 
and  so  preparing  the  way  for  the  formation  of  pus.  It  is 
still  doubtful  how  much  reliance  can  be  placed  on  the 
total  separation  of  the  soluble  and  endotoxins  from  the 
bacterial  protein,  and  until  this  doubt  is  resolved,  these 
conclusions  must  be  accepted  with  reserve. 

Extracellular  Toxins.  —  Of  the  extracellular  or  true 
or  soluble  toxins,  that  of  B.  diphtheriae  may  be  taken  as 
the  type.  As  a  class  they  may  be  defined  as  the  secretory 
products  of  the  bacterial  cells,  passing  out  into  the  medium, 
and  soluble  therein.  That  the  soluble  toxins  are  only  so 
produced  has  yet  to  be  proved,  and  in  the  meantime  they 
may  be  ascribed  to  the  following  sources,  which  may  occur 
singly,  or  in  any  combination  : — 

i.  vSecretion  or  excretion  from  the  bacterium. 

2.  Action  of  the  bacterium  on  the  medium. 

3.  Death  of   the  bacterium  and  liberation  of  toxins 

from  its  disintegrated  body. 

The  soluble  toxins  are  easily  obtainable  in  large 
quantities,  nevertheless  they  have  not  yet  been  isolated 
in  a  pure  form,  and  so  our  knowledge  of  them  is  derived 
from  the  study  of  the  complex  filtrates  in  which  they  are 
found.  Their  action  is  characterized  by  being  selective 
or  specific  for  certain  tissues ;  for  example,  diphtheria, 
tetanus,  and  botulismus  toxins  all  attack  the  nervous 
system.  In  the  case  of  many  of  them,  a  definite  time 
elapses  before  symptoms  appear  after  injection.  This 
has  been  called  a  period  of  incubation,  though  it  is  suscep- 
tible of  other  explanations.  The  extracellular  toxins  are 
apparently  uncrystallizable,  are  soluble  in  water,  are  dia- 
lysable,  are  precipitated  with  proteids  by  absolute  alcohol 
and  by  ammonium  sulphate,  are  allied  to  albumoses,  and 
are  relatively  unstable  to  heat,  light,  and  chemicals. 

Intracellular  Toxins.  —  The  endotoxins  are  either 
not  excreted  from  the  bodies  of  the  bacilli,  or  are  closely 
bound  thereto,  or  are  insoluble  in  the  medium,  and  remain 
on  filtration  on  the  same  side  as  the  bacteria.  Any  of 
these  theories  would  account  for  the  known  facts  in  regard 
to  the  endotoxins.  The  greater  number  of  the  pathogenic 
bacteria  seem  to  act  by  poisons  of  this  class.     The  poisons 


BACTERIAL    ACTIVITY  177 

are  Jiberated  only  after  the  death  of  the  bacteria,  by  the 
breaking  up  of  their  bodies,  and  even  then  they  cannot 
be  obtained  apart  from  the  bacterial  protoplasm.  Their 
action  has  therefore  been  mostly  studied  by  injection  of 
the  dead  bodies  of  bacteria.  The  effects  produced  are 
not  specific,  but  are  more  those  of  general  disturbances 
of  metabolism ;  nor  does  much  time  elapse  before  the 
appearance  of  the  symptoms,  that  is,  there  is  no  so-called 
incubation  time. 

The  intracellular  toxins  are  less  sensitive  to  heat  than 
the  soluble  ones,  but  are  mostly  destroyed  by  heating  to 
700  C.  The  notable  exceptions  to  this  are  those  of  the 
tubercle  bacillus,  which  are  still  toxic  after  digestion  at 
ioo°  C,  and  those  of  the  B.  enteritidis  (Gaertner)  which 
remain  toxic  after  the  infected  flesh  has  been  cooked. 

Some  organisms,  such  as  B.  anthracis,  possess  no  soluble 
toxin,  nor  does  the  injection  of  the  dead  bodies  induce 
toxic  effects.  Yet  in  the  disease  produced  by  the  living 
bacterium,  symptoms  which  suggest  toxin  action  are 
present.  To  meet  such  cases,  the  hypothesis  has  been 
made  that  these  organisms  only  produce  toxins  in  the 
animal  tissues,  or  may  produce  complementary  substances 
which  assist  the  action  of  endotoxins.  Such  substances 
have  been  studied  under  the  name  of  "  aggressins."  An 
animal  is  given  a  lethal  dose  of  an  organism,  injected  into 
a  serous  cavity,  into  which  a  serous  exudation  results.  On 
the  death  of  the  animal,  some  of  the  exudate  is  taken, 
most  of  the  bacteria  are  removed  by  centrifugalizing,  and 
the  few  that  remain  by  shaking  up  with  toluol,  and  allowing 
to  stand  for  some  days.  This  fluid,  on  injection,  is  non- 
pathogenic, but  has  the  power  of  increasing  the  effect 
of  the  particular  bacterium  which  has  caused  its  production, 
so  that  a  non-lethal  dose  of  the  bacterium  becomes  a 
lethal  one ;  and  not  only  so,  but  the  fatal  effect  is  more 
quickly  produced.  These  results  are  ascribed  to  a 
paralysing  action  of  the  "  aggressins  "  on  the  phagocytic 
functions  of  the  leucocytes.  Leucocidin,  a  true  soluble 
toxin  produced  by  some  strains  of  staphylococcus,  causes 
the  death  and  partial  solution  of  the  leucocytes,  and  this 
would  suggest  that  the  aggressins  might  after  all  be  toxins, 
either  of  the  extra-  or  intra-cellular  variety. 

12 


178         PUBLIC    HEALTH    BACTERIOLOGY 

Some  bacteria  give  rise  to  both  varieties,  and  it  is  now 
claimed  that  this  is  the  case  with  cholera  and  dysentery 
organisms. 

Nature  of  Toxins. — The  nature  of  toxins  is  likewise 
ill  understood.  Sidney  Martin  found  that  the  action  of 
anthrax,  diphtheria,  tetanus,  and  ulcerative  endocarditis 
organisms  on  albuminous  bodies  was  to  produce  albumoses 
and  peptones,  thus  resembling  the  gastric  and  pancreatic 
ferments.  C.  J.  Martin,  working  at  the  same  subject, 
found  that  the  toxins  could  pass  through  a  Chamberland 
filter,  the  pores  of  which  had  been  filled  with  gelatin. 
From  the  fact  that  albumoses  can  also  pass  through,  it 
is  inferred  that  the  toxins  have  a  molecule  of  about  the 
same  size  as  the  albumoses.  Are  the  toxins  of  the  nature 
of  ferments  ?  Sidney  Martin  suggests  that  the  primary 
toxic  agents  are  of  this  nature,  and  by  digesting  the  tissues 
produce  albumoses,  which  cause  the  symptoms.  The 
labile  nature  of  the  toxins  is  also  urged  as  a  point  of 
resemblance  between  them  and  the  ferments,  as  also  the 
so-called  period  of  incubation  which  follows  their  injection. 
If  it  is  a  fact  that  the  action  of  a  toxin  is  strictly  propor- 
tional to  its  dose,  comparison  between  toxins  and  the 
ferments  is  rendered  unnecessary,  as  this  is  a  fundamental 
difference  ;  the  so-called  resemblances  are  then  mainly 
fortuitous. 

Allied  Animal  and  Vegetable  Poisons.  —  Ricin, 
abrin,  robin,  and  venins.  Major  Lamb  calculates  that 
0-015  grm-  (roughly,  J  grain)  of  cobra  venom  is  a  fatal 
dose  for  a  man,  which  is  large  in  comparison  with  the 
minimum  lethal  dose  of  tetanus  toxin  for  man  of  0-00023 
grm.  (about  ^-^  grain).  All  these  poisons  resemble  the 
soluble  bacterial  toxins,  but  are  less  easily  dialysable,  and 
hence  have  been  called  toxalbumins.  The  snake  poisons 
are  very  complex  bodies,  containing  one  or  more  of 
several  toxins,  such  as  neurotoxins,  cell  toxins,  hemolytic 
toxins,  etc. 

Flexner  and  Noguchi  discovered  that  the  hemolytic 
toxin  of  the  cobra  venom  has  no  action  by  itself  on  the 
red  cells,  but  requires  the  presence  of  normal  serum. 
The  latter  is  then  said  to  contain  a  "  complement  "  which 
"activates"  the  venom.     Kyes  and  Sachs  farther  showed 


BACTERIAL    ACTIVITY  179 

that  lecithin  (a  highly  complex  fat  found  in  the  nervous 
system,  and  to  a  less  extent  in  bile)  has  the  property  of 
activating  the  haemolytic  substance  in  cobra  venom.  This 
is  very  important,  since  it  points  to  a  definite  chemical 
combination,  leading  to  the  formation  of  a  toxin  from  two 
non-toxic  bodies,  and  is  in  line  with  the  observed  for- 
mation in  diphtheria  of  an  antitoxin,  which  has  many  of 
the  characters  of  a  chemical  antidote. 


CHAPTER    XL 
IMMUNITY     AND     ANAPHYLAXIS. 

Immunity,  or  resistance,  may  be  denned  as  that  power 
or  function  of  the  living  organism,  natural  or  acquired, 
which  enables  it  to  repel  or  prevent  infection  of  itself  by 
micro-organisms  or  their  products. 

Anaphylaxis,  or  excessive  susceptibility  (hypersuscep- 
tibility,  supersensitiveness),  is  defined  as  a  state  of 
extreme  sensitiveness  to  the  injection  of  certain  substances, 
such  as  bacterial  proteins,  animal  and  vegetable  albumins 
(blood  serum,  egg  white,  milk),  brought  about  by  one 
injection  of  the  same  substances  or  present  from  hereditary 
transmission. 

Both  these  terms  are  relative,  in  most  instances.  Thus, 
birds,  while  immune  from  tetanus  toxin  in  any  doses 
likely  to  result  from  natural  infection,  may  be  killed  by 
enormous  doses  given  experimentally.  Similarly,  in  man, 
the  immunity  conferred  by  one  attack  of  a  disease  like 
small-pox,  may  be  overcome  in  special  circumstances  of 
dosage  and  environment. 

Absolute  immunity  does  exist.  Thus,  so  far,  no  animal 
has  been  infected  with  leprosy  ;  also,  cold-blooded  animals, 
under  their  normal  conditions,  are  absolutely  immune  to 
the  pathogenic  bacteria  of  the  warm-blooded  animals. 
The  wild  carnivora  have  a  very  high  degree  of  resistance 
to  bacteria. 

Absolute  anaphylaxis  of  a  kind  also  occurs.  Thus,  the 
injection  (subcutaneously)  of  0-25  c.c.  of  the  serum  of  an 
eel  into  a  rabbit  causes  the  death  of  the  rabbit  in  a  few 
minutes.  Also  the  offspring  of  animals  which  have  been 
themselves  sensitized  by  injection,  show  a  high  degree  of 
anaphylaxis  from  birth.  (The  strict  use  of  the  term 
"  absolute  "  would  require  that  the  rabbit  should  die  no 
matter  how  small  the  dose  of  serum  used.  Thus  its  use 
here  is  relative.) 


IMMUNITY    AND    ANAPHYLAXIS  181 

IMMUNITY. 

Immunity  may  be  natural  or  acquired,  and  may  be 
considered  under  the  following  heads: — 

Natural  Immunity. — Depends  on  (a)  individual,  (b)  race, 
and  (c)  species. 

Acquired  Immunity. — (a)  By  an  attack  of  the  specific 
disease  ;  (b)  By  active  immunization  with  living  bacteria, 
dead  bacteria,  or  with  toxins  or  filtrates  ;  (c)  By  passive 
immunization  with  antitoxic  serum,  or  antibacterial  serum. 

Natural  Immunity,  or  the  resistance  to  bacteria  con- 
ferred by  nature,  is  a  characteristic  of  the  living  organism, 
which  varies  with  the  individual,  race,  and  species.  Thus, 
individuals  vary  greatly  in  their  resistance  to  infection 
from  slight  wounds,  from  polluted  water  and  milk,  and 
from  micro-organisms  generally.  The  young  animal  is 
usually  less  resistant  than  the  mature  of  the  same  kind. 
As  regards  race,  negroes  are  noted  for  their  high  degree  of 
resistance  to  yellow  fever,  and  in  a  less  degree  to  malaria, 
yet  they  quickly  sicken  and  succumb  to  small-pox  and 
measles.  Among  animals,  the  Algerian  sheep  are  more 
highly  resistant  to  anthrax  than  the  European  races. 
The  influence  of  species  is  seen  in  the  non-liability  of  the 
human  to  certain  animal  diseases,  such  as  cattle  plague, 
fowl  cholera,  and  swine  erysipelas,  whilst  animals  are 
equally  resistant  to  such  human  infections  as  cholera, 
influenza,  measles,  etc. 

The  causes  of  natural  immunity  are  usually  given  as : — 
(i)  The  action  of  certain  leucocytes  and  other  cells  in 
engulfing  and  destroying  the  bacterial  invaders, 
called  phagocytosis,  and 
(2)  The  action  of  the  blood  serum. 

I.  Phagocytosis. — Metchnikoff  in  1884  advanced  the 
theory  of  phagocytosis,  based  on  a  careful  study  of  the 
subject,  and  since  confirmed  by  many  observers.  The 
phagocytes  are  in  part  wandering  cells,  and  in  part  fixed 
tissue  cells.  The  chief  wandering  cells  are  the  poly- 
morphonuclear and  large  mononuclear  leucocytes  and 
wandering  tissue  cells.  Of  the  fixed  phagocytes,  possessing 
the  power  of  amoeboid  movement,  the  cells  lining  the  serous 
and  lymph  spaces,  the  cells  of  the  spleen  pulp,  and  bone 


182         PUBLIC    HEALTH    BACTERIOLOGY 

marrow,  are  the  chief  examples.  Metchnikoff  calls  the  poly- 
morphonuclear leucocytes,  Microphages,  and  all  the  other 
phagocytes,  Macrophages.  He  observed  the  phagocytosis 
in  a  fungus  disease  of  a  water-flea  (Daphnia),  and  in  frogs 
infected  with  anthrax,  where  the  death  and  dissolution  of 
the  bacilli  could  be  seen  going  on  inside  the  phagocytes. 
Later  researches  proved  that  the  same  phenomena  could 
be  observed  in  all  infective  processes,  more  especially  if 
the  animal  were  resistant  to  the  infection.  When  the 
micro-organisms  get  into  a  part  where  few  phagocytes 
are,  a  migration  towards  the  affected  spot  occurs.  This 
is  part  of  the  inflammatory  reaction  which  follows  infection. 
The  cause  of  this  migration  is  the  presence  of  substances 
in  the  part,  which  attract  the  phagocytes,  and  is  known 
as  positive  chemiotaxis.  Negative  chemiotaxis,  or  the 
repulsion  of  the  phagocytes,  also  occurs.  Buchner  showed 
that  dead  bacteria,  bacterial  proteins,  and  closely  allied 
substances,  such  as  vegetable  casein  (legumin),  have  a 
positive  Chemiotaxis,  whilst  the  toxins  of  many  virulent 
bacteria  have  a  negative  chemiotactic  power.  In  natural 
immunity,  phagocytosis  is  developed  to  a  high  degree, 
and  it  is  of  such  constant  and  regular  occurrence  that 
we  may  often  foretell  from  the  degree  of  phagocytosis 
whether,  in  a  particular  infection,  the  animal  being 
experimented  with  will  gain  the  victory  or  not.  A  clinical 
application  of  these  results  is  seen  in  the  observation  of 
the  increase  of  the  number  of  leucocytes  in  the  blood 
during  the  progress  of  a  disease  like  pneumonia.  The 
increase  is  called  "  Leucocytosis,"  and  is  almost  wholly 
of  the  polymorphonuclear  variety.  In  pneumonia  an  early 
and  marked  leucocytosis  is  a  sign  of  favourable  import, 
and  may  be  from  12,000  to  40,000  per  c.mm.  The  absence 
of  leucocytosis,  except  in  very  slight  infections,  is  highly 
unfavourable.  (It  is  interesting  to  note  here  that  whoop- 
ing-cough gives  a  Lymphocytosis,  as  also  do  enlarged 
tonsils,  rickets,  scurvy,  and  a  few  other  diseases.) 

2.  The  Action  of  the  Blood  Serum. — Besides  the 
direct  action  of  the  phagocytes,  as  described  by  Metchnikoff 
(the  cellular  theory),  the  blood  seium  was  found  to  have 
bactericidal  power.  Von  Fodor  showed  that  freshly 
drawn  rabbit's  blood  could  destroy  anthrax  bacilli,   as 


IMMUNITY    AND    ANAPHYLAXIS  183 

also  could  defibrinated  blood,  the  pericardial  fluid  and 
the  aqueous  humour,  and  that  this  power  was  lost  by 
heating  to  550  C.  Buchner  (with  others)  found  that 
completely  cell-free  blood  was  bactericidal,  and  lost  this 
power  on  heating  to  550  C,  but  not  on  freezing  and 
thawing.  According  to  Buchner,  fresh  blood  frozen  and 
thawed  loses  its  power  because  the  red  cells  are  destroyed 
by  the  process,  and  make  the  blood  so  suitable  for 
bacteria  that  the  bactericidal  power  is  compensated  for. 
These  protective  substances  in  the  serum  are  called 
"  Cytases  "  by  Metchnikoff,  and  "Alexines  "  by  Buchner. 
These  are  now  believed  to  be  derived  from  the  leucocytes, 
Metchnikoff  holding  that  they  are  only  formed  on  the 
death  of  the  leucocytes,  or  rather  phagocytes  ("  phago- 
lysis"),  and  that  they  do  not  exist  in  the  body  except 
under  abnormal  conditions.  In  any  case  when  present  they 
will  probably  prepare  the  bacteria  for  ingestion  by  the 
phagocytes,  and  thus  are  related  to,  if  not  identical  with 
the  Opsonins  "  (feast-preparers).  The  cytases  or  alex- 
ines are  of  proteid  nature  and  are  very  unstable.  The 
withdrawal  of  the  salts  from  the  serum  by  dialysis  sus- 
pends their  activity,  which  is  restored  on  again  adding 
them.  This  fact  is  evidently  related  to  the  lessened 
amount  of  chlorides  excreted  in  the  urine  in  all  acute 
febrile  processes,  and  especially  in  lobar  pneumonia. 
The  cytases  or  alexines  may  be  regarded  as  an  appliance 
common  to  every  animal  organism,  for  the  dissolution  of 
organized  substances,  whether  bacteria,  foreign  red 
corpuscles,  or  other  foreign  bodies.  They  are  the 
"  Complements"  of  Ehrlich's  classification. 

Acquired  Immunity. — The  immunity  that  is  called 
natural  is  of  a  general  kind,  being  a  natural  resistance  to 
disease  or  bacteria  of  all  kinds.  That  type  of  immunity 
called  acquired,  is,  on  the  other  hand,  "specific"  in  kind, 
that  is,  an  immunity  from  a  definite  or  specific  disease  or 
infection.  For  this  reason  it  is  by  some  called  specific 
immunity. 

(a).  Acquired  by  an  attack  of  a  Specific  Disease. — The 
fact  that  an  attack  of  small-pox  followed  by  recovery 
protected  the  individual  from  further  attack,  was  a  notable 
one  during  the  epidemic  prevalence  of  that  disease.  Similar 


184         PUBLIC    HEALTH    BACTERIOLOGY 

protection  was  seen  in  regard  to  other  eruptive  and 
non-eruptive  fevers  such  as  scarlatina,  measles,  typhus, 
typhoid,  and  whooping-cough.  On  the  contrary,  some 
specific  diseases  do  not  protect,  but  one  attack  seems  to 
render  the  individual  more  liable  to  another.  Such  are 
diphtheria,  pneumonia,  influenza,  gonorrhoea,  erysipelas, 
relapsing  fever,  and  rheumatic  fever.  In  all  of  these  it  is 
probable  that  some  degree  of  immunity  results,  but  is  of 
very  short  duration,  as  has  been  definitely  observed  in 
cholera  and  some  other  diseases.  In  all  cases,  however 
short  the  immunity,  it  is  absolutely  specific  against  a 
certain  infective  agent  or  its  poison,  and  is  not  due  to  a 
general  increase  of  resistance.  In  fact  the  reduction  of 
the  general  resistance  following  the  specific  infection  is 
such  as  in  some  cases  to  predispose  to  other  infections. 
Thus,  tuberculosis  not  infrequently  follows  a  severe 
attack  of  measles,  whooping-cough,  or  typhoid.  Recovery 
from  an  acute  infective  disease  is  due  to  a  process  of 
immunization  going  on  during  the  progress  *of  the  disease, 
which  at  a  certain  point  or  stage  is  able  to  prevent  the 
further  action  of  the  infecting  agent.  The  substances 
formed  do  not  always  exterminate  the  virus  from  the 
mucous  surfaces ;  and  this  is  seen  specially  in  typhoid 
fever,  where  the  recovered  patient  may  continue  to  excrete 
the  living  virus  by  the  bowel  or  urinary  discharges,  and 
in  diphtheria,  where  the  virus  may  persist  in  the  throat. 

Artificial  Immunity. — Under  this  head  may  be  classed 
together  forms  (b)  and  (c)  of  acquired  immunity. 

(b).  By  Active  Immunization,  or  Protective  Inoculation— 
where  the  specific  protective  substances  have  to  be  formed 
in  the  body  itself,  as  opposed  to  immunization  by  trans- 
ference of  protective  substances  formed  by  active 
immunization  in  another  animal,  and  called  passive 
immunization.  In  active  immunization,  the  individual  or 
animal  must  undergo  an  infection  followed  by  a  reaction. 
By  this  means  the  protective  substances  are  formed,  and 
so  the  immunity  is  obtained  only  after  the  lapse  of  a  period 
of  time,  when  the  immunizing  apparatus  of  the  organism 
is  able  to  produce  the  protective  substances  in  sufficient 
amount.  The  immunity  thus  evoked  is  of  a  more 
persistent  type  than  that  obtained  by  simply  transferring 


IMMUNITY    AND    ANAPHYLAXIS  185 

the  protective  substances  from  an  immunized  animal, 
because,  in  the  first  case,  an  immunizing  apparatus  has 
been  set  up  which  is  able  to  produce  an  (apparently) 
indefinite  amount  of  protective  substances  and  over  a 
long  period,  whereas  in  the  second  case,  a  definite  amount 
of  the  protective  substances  is  injected,  and  when  this 
amount  is  used  up  the  protection  is  at  an  end.  In  active 
immunization,  we  must  distinguish  a  "  specific  immunity 
to  bacteria,"  and  a  "  specific  immunity  to  their  toxins," 
just  as  we  have  a  natural  resistance  or  immunity  to 
bacteria,  which  is  different  from  the  natural  resistance  or 
immunity  to  certain  poisons.  Thus  the  immunity  after 
diphtheria  is  mainly  to  the  toxins  (antitoxic).  On  the 
other  hand,  the  immunity  purchased  by  the  injection  of 
cholera  vibrios,  is  merely  to  the  bacteria  and  not  to  their 
endotoxins.  Hence  the  injection  of  cholera  spirilla  into  an 
animal  previously  immunized  to  the  same,  is  followed  by 
the  death  and  dissolution  of  the  spirilla,  but  if  the  dose  is 
large  enough,  also  by  a  fatal  intoxication  of  the  animal 
by  the  cell  poisons  thus  suddenly  set  free.  This  is  the  chief 
cause  of  failure  of  immunization  to  those  bacteria  which 
do  not  act  (as  diphtheria  and  tetanus  do)  through  soluble 
toxins,  diffused  into  the  blood  stream.  The  immunization 
would  require,  in  such  cases,  to  be  of  a  double  nature, 
namely,  antibacterial  and  antitoxic.  Apparently,  as 
usually  induced,  the  former  mainly,  if  not  entirely,  results. 
The  methods  of  active  immunization  are  based  on  the 
work  and  discoveries  of  Pasteur  and  his  associates,  and 
may  be  summarized  thus : — 

(i)  With  living  bacteria,  virulent  or  attenuated. 

(2)  With  dead  bacteria. 

(3)  With  the  bacterial  cell  substances. 

(4)  With  soluble  toxins  or  filtrates. 

(5)  By  feeding  with  toxic  substances. 

1.  With  Living  Bacteria  or  Virus,  Virulent  or  Attenuated. 
— (a).  With  virulent  virus.  Though  the  virus  of  small-pox 
is  still  unknown  with  certainty,  inoculation  of  the  small- 
pox, as  introduced  into  England  in  171 8  by  Lady  Mary 
Wortley  Montagu  (see  her  "  Letters  "),  may  be  given  as  an 
example.    The  inoculated  disease  was  usually  mild  in  type, 


186         PUBLIC    HEALTH    BACTERIOLOGY 

but  at  times  was  severe  and  even  fatal.  In  contagious 
pleuro-pneumonia  of  cattle,  subcutaneous  injection  of  the 
lymph  of  a  newly  killed  animal,  into  the  tail,  has  proved 
protective.  The  animal  sometimes  loses  its  tail  in  part, 
the  brunt  of  the  infection  apparently  remaining  localized. 

(b).  With  attenuated  virus.  This  is  accomplished  in 
one  or  more  of  the  following  ways  :— 

By  cultivation  of  the  organism  in  oxygen  or  a  current  of 
air,  as  first  discovered  by  Pasteur  in  the  case  of  the  bacilli 
of  chicken  cholera.  The  attenuated  bacilli,  on  injection, 
produced  a  non-fatal  disease,  which  immunized  the  fowl, 
especially  on  repetition. 

By  cultivation  of  the  bacteria  at  high  temperatures, 
e.g.,  anthrax  bacilli  at  420  to  430  C. 

By  passage  through  a  less  susceptible  species.  This  is 
the  presently  accepted  explanation  of  vaccination  for 
small-pox.  Used  by  Pasteur  for  swine  erysipelas,  the 
bacillus  of  which  is  lessened  in  virulence  by  repeated 
passage  through  rabbits,  but  increased  by  passage  through 
pigeons.  Two  inoculations  were  given,  the  first  of  the 
attenuated  bacilli  from  rabbits,  the  second  of  the  exalted 
bacilli  from  pigeons. 

By  drying  the  virus,  as  in  hydrophobia,  where  Pasteur 
found  that  the  virus  was  exalted  in  virulence  by  successive 
subdural  passage  through  rabbits,  but  diminished  by 
passage  through  apes.  He  tried  the  immunization  of  dogs 
by  the  use  of  the  diminished  and  increased  viruses,  but 
the  results  were  too  variable,  so  he  tried  drying  the  spinal 
cord  of  a  rabbit  dead  of  the  disease.  A  cord  thus  dried  at 
220  C.  over  KOH  (to  absorb  C02)  for  1  to  4  days,  still 
causes  rabies  in  7  days  (the  incubation  period  shortened 
from  14  days  by  the  exaltation  of  the  virus) ;  but  if  kept 
longer,  the  incubation  period  is  prolonged,  until  one  kept 
12  to  14  days  has  become  inactive.  The  treatment 
consists  in  beginning  with  injection  of  an  emulsion  of  this 
cord,  and  daily  repeating  with  an  emulsion  of  a  less  dried 
cord  until  within  15  days  in  mild  cases  and  21  days  in 
severe,  the  strength  arrived  at  is  a  3-day-dried  cord. 
Complete  immunity  thus  takes  3  to  4  weeks,  and  so  in 
cases  coming  under  treatment  at  a  late  period,  the 
method  is  condensed.     Hoegyes  uses  fresh  cord  emulsified 


IMMUNITY    AND    ANAPHYLAXIS  187 

in  salt  solution,  of  which  dilutions  are  made,  and  injections 
made  in  reverse  order  of  dilutions.  Fewer  accompanying 
symptoms  (as  erythema  at  the  point  of  injection,  backache, 
muscular  pains,  and  occasionally  temporary  paralysis) 
are  noted,  and  this  result  is  attributed  to  the  less  amount 
of  nerve  tissue  injected. 

By  cultivation  in  a  medium  containing  antiseptics  in 
a  dilute  state  ;  e.g.,  in  presence  of  carbolic  acid  1-600,  or 
potassium  bichromate  1-5000,  or  sulphuric  acid  1-200. 

By  addition  of  weak  antiseptic  solutions  to  virulent 
broth-cultures  preparatory  to  their  injection,  as  advised 
by  Behring  for  immunization  of  horses  to  diphtheria  and 
tetanus.  The  antiseptic  advised  is  iodine  terchloride, 
ICI3,  in  strengths  varying  from  0*05  per  cent  to  0-4  per 
cent.     Lugol's  solution  of  iodine  is  also  used. 

2.  With  Dead  Bacteria. — This  method  is  simpler  and 
safer,  and  in  many  cases  confers  the  same  degree  of 
immunity,  which  is  chiefly  antibacterial.  It  is  also 
used  preliminarily  to  injection  of  living  cultures,  in  the 
active  immunization  of  animals.  In  the  human  being, 
it  is  used  for  cholera,  plague,  typhoid,  and  in  the  treat- 
ment by  "  vaccines "  generally,  as  for  staphylococcus 
infection,  etc. 

3.  With  Bacterial  Cell  Substances. — This  method  is  a 
modification  of  that  with  dead  bacteria.  Instead  of 
injecting  the  culture  heated  to  650  C,  or  some  such  tempera- 
ture, to  kill  the  bacteria,  the  culture  is  subjected  to  various 
processes  as  in  the  preparation  of  Koch's  original  tuberculin. 
This  is  not  purely  bacterial  cell  substances,  but  is  inter- 
mediate between  the  simply  heated  culture  and  tuberculin- 
R,  which  is  an  emulsion  of  the  bodies  of  bacilli  from  which 
all  the  soluble  substances  have  been  extracted  by  grinding 
and  treatment  with  distilled  water  (tuberculin-O).  Hahn, 
following  Buchner,  has  used  mechanical  pulverization  of 
bacilli  mixed  with  infusorial  earth  and  quartz  sand,  and 
subjected  to  300  to  500  atmospheres'  pressure  by  hydraulic 
means,  and  has  so  obtained  what  he  calls  the  cell-juices 
or  bacterial  plasmins,  which  he  has  used  for  immunization. 
"  Cholera  plasmin  "  and  "  typhoid  plasmin  "  have  both 
proved  effective  in  immunizing  guinea-pigs  against  intra- 
peritoneal infection  with  ten  times  the  fatal  dose  of  virulent 


188         PUBLIC    HEALTH    BACTERIOLOGY 

bacteria.  "  Tuber culo-plasmin  "  after  filtration  is  a  clear 
pale  yellow  fluid,  containing  nucleo-albumin,  which  keeps 
indefinitely  on  addition  of  20  per  cent  of  glycerin  and 
5  per  cent  of  NaCl.  This  preparation  has  been  used  with 
favourable  results  in  guinea-pig  tuberculosis. 

The  reaction  which  follows  the  injection  of  a  dead 
culture  (local  pain  and  swelling,  rigor,  depression,  and 
anorexia)  is  not  peculiar  to  any  one  bacterium,  but 
follows  upon  the  subcutaneous  injection  of  all  bacterial 
emulsions,  and  even  of  innocuous  and  living  bacteria 
(Buchner,  1890). 

4.  With  Soluble  Toxins  or  Filtrates. — This  method  was 
first  successfully  used  by  Salmon  and  Smith,  who  showed 
that  pigeons  could  be  rendered  immune  to  hog  cholera 
by  treatment  with  filtrates  of  hog-cholera  bacilli  (1886). 
It  is  now  used  for  the  immunization  of  horses  to  diphtheria 
and  tetanus  toxins,  the  immunity  being  afterwards 
heightened  by  the  injection  of  virulent  cultures,  if  Behring's 
advice  is  adopted.  This  use  followed  on  the  observations 
of  Roux  and  Yersin,  who  showed  in  1886,  as  a  "  result 
of  splendid  research "  (Buchner)  that  the  poison  of 
diphtheria  is  extremely  susceptible  to  heat  (being  destroyed 
at  650  C),  and  is  carried  down  mechanically  by  chemical 
precipitates  such  as  calcium  phosphates,  properties  which 
until  then  had  been  recognized  mainly  in  the  digestive 
ferments  or  enzymes.  Brieger  and  Fraenkel  confirmed 
these  results,  and  showed  that  the  poisons  or  toxins  of 
diphtheria  and  tetanus  can  be  obtained  in  a  moderately 
pure  form  by  precipitation  with  absolute  alcohol.  These 
poisons  gave  the  reactions  of  albuminous  substances, 
and  were  hence  at  first  called  "  toxalbumins."  As  they 
are  now  believed  to  be  non-proteid,  the  name  "  specific 
toxins  "  is  to  be  preferred.  A  specific  toxin  is  one  which, 
on  injection,  causes  all  the  symptoms  of  the  infection  in 
question. 

5.  Active  Immunization  by  Feeding  has  been  successfully 
used  by  Ehrlich  for  the  poisons  ricin  and  abrin,  and  with 
less  success  by  Fraser  against  snake  venom.  In  bacterial 
infections  it  has  proved,  so  far,  tedious,  and  the  immuni- 
zation slight  in  amount. 

(c).  By     Passive    Immunization.  —  Behring    in     1890 


IMMUNITY    AND    ANAPHYLAXIS  189 

discovered  that  the  blood  serum  of  an  animal  actively 
immunized  against  diphtheria,  when  injected  into  another 
animal,  is  capable  of  rendering  the  latter  insusceptible  to 
what  would  otherwise  be  a  fatal  dose  of  diphtheria  toxin. 
That  is,  the  serum  was  able  to  destroy  the  diphtheria 
poison.  In  conjunction  with  Kitasato  he  afterwards  proved 
the  same  for  the  tetanus  toxin.  The  transference  of  the 
immune  serum,  in  both  cases,  protects  against  the  specific 
poison,  and  so  allows  the  natural  resistance  of  the  body 
to  overcome  the  bacilli.  As  the  new  animal  body  has 
thus  supplied  to  it  an  antidote  to  the  microbic  toxins 
which  have  been  shown  to  produce  the  symptoms  of  the 
disease,  no  reaction  to  these  toxins  occurs  (where  they 
have  been  completely  and  early  destroyed),  and  so  no 
active  immunization  occurs.  The  immunity  conferred  is, 
therefore,  of  a  transient  nature,  and  lasts  only  as  long  as 
some  of  the  immune  body  in  excess  persists  in  the  blood. 
Any  such  excess  is  destroyed  or  excreted  within  eight  to 
fourteen  days,  and  so  the  immunity  may  be  expected  to 
be  absent  thereafter.  These  remarks  apply  to  "  antitoxic 
sera,"  like  those  obtained  in  diphtheria  and  tetanus.  The 
other  form  of  passive  immunization  by  "  antibacterial " 
or  "  antimicrobic  "  sera,  has  proved  unsatisfactory  in  use, 
for  the  reasons  given  on  page  185.  The  action  of  the  latter 
sera  is  not  so  simple  as  that  of  antitoxic  sera,  but  is  due 
to  one  or  more  of  the  following  factors  :  (1)  Bactericidal 
or  lysogenic  action,  that  is,  death  or  solution  of  the  micro- 
organisms ;  (2)  Opsonic  action,  or  the  rendering  the 
bacteria  more  susceptible  to  the  phagocytic  action  of  the 
leucocytes ;  (3)  Agglutinative  and  precipitative  actions, 
that  is,  clumping  of  the  bacteria,  or  precipitation  of  their 
soluble  products. 

Antitoxic  Sera. — The  actual  mode  of  manufacture 
may  be  conveniently  given  here.  Diphtheria  antitoxin 
may  be  taken  as  the  type.  A  culture  of  B.  diphtherial  in 
meat-infusion  broth  (containing  1  to  2  per  cent  of  added 
peptones,  and  after  being  made  neutral  to  litmus,  having 
7  c.c.  of  N/i  NaOH  added  per  litre)  is  incubated  for  three 
weeks  at  370  C.  A  strongly  toxic  fluid  is  thus  produced, 
which  is  filtered  through  a  Chamberland  candle  into  a  sterile 
flask,  care  being  taken  to  avoid  exposure  to  bright  light. 


190         PUBLIC    HEALTH    BACTERIOLOGY 

At  the  first  attempts  at  immunization,  the  animals  died 
of  chronic  poisoning.  To  avoid  this,  it  is  now  usual  to 
begin  with  very  small  doses  of  the  toxin  weakened  by  the 
addition  of  iodine  terchloride  or  Lugol's  solution  (used  in 
Gram's  method  of  staining,  I  in  KI  in  water).  A  young 
vigorous  healthy  horse  (4  to  6  years  old)  is  chosen,  and 
0-5  c.c.  of  toxin  mixed  with  an  equal  quantity  of  Lugol's 
solution  is  injected  subcutaneously  in  front  of  the  shoulder- 
blade,  using  a  large  needle  connected  with  a  syringe  by  a 
piece  of  rubber  tubing.  (The  skin  is  previously  shaved 
and  sterilized.)  After  the  reaction  has  subsided,  usually 
in  5  to  8  days,  a  few  days'  interval  is  given,  and  the  next 
dose  is  administered,  either  1  c.c.  -j-  1  c.c.  Lugol's  solution 
or  05  c.c.  pure  toxin.  The  amount  given  is  thus  gradually 
increased  by  \  c.c.  until  finally  in  three  to  four  months 
the  antitoxic  value  of  the  animal's  serum  is  such  that  a 
dose  of  300  c.c.  of  active  toxin  may  be  borne.  The 
immunizing  process  must  not  be  pushed  too  rapidly,  other- 
wise the  health  of  the  animal  will  suffer.  Serum  is  then 
obtained  by  inserting  a  sterile  needle  into  the  jugular  vein 
of  the  horse,  and  collecting  the  quantity  of  blood  desired 
(up  to  6  litres  at  a  time)  through  a  rubber  tube  into  sterile 
Erlenmeyer  flasks  containing  solution  of  citrate  of  soda. 
These  are  allowed  to  stand  until  the  corpuscles  settle,  and 
the  plasma  is  poured  off  into  another  flask,  and  allowed 
to  clot.  Separate  the  serum,  and  filter.  If  filtered  at 
once,  it  is  found  to  precipitate  again  on  standing,  hence 
it  is  better  to  allow  it  to  stand  a  few  days  before  filtration. 
Bottle  or  tube,  after  adding  05  per  cent  carbolic,  but 
before  this  it  should  be  standardized. 

Standardization  of  antitoxic  serum  is  very  important, 
so  that  accurate  dosage  may  be  determined,  and  also  so 
that  one  strength  may  be  aimed  at,  since  in  the  making 
it  is  liable  to  variation.  The  making  of  such  a  standard  is 
not  an  easy  task,  because  no  two  samples  of  toxin  are  of 
exactly  the  same  strength,  nor  are  even  two  samples  of  the 
same  toxin  tested  at  different  times.  This  is  another  way 
of  saying  that  toxin  is  a  very  variable  substance,  but 
fortunately  antitoxin  is  not  so  variable.  Hence  Ehrlich 
chose  as  his  "  immunity  unit,"  the  amount  of  antitoxic 
serum  which  will  neutralize  100  times  the  minimum  lethal 


IMMUNITY    AND    ANAPHYLAXIS  191 

dose  (M.L.D.)  of  toxin  for  one  guinea-pig  of  250  grm.  weight 
and  which  kills  it  within  5  days ;  the  serum  and  toxin 
being  mixed  together  and  made  up  to  4  c.c.  bulk,  and 
injected  subcutaneously,  the  animal  survives  the  time- 
limit.  A  serum  containing  one  such  unit  in  1  c.c.  is 
called  "normal  serum"  or  "normal  diphtheria  antitoxin" 
(D.A.N.),  while  a  serum  1  c.c.  =  1,000  M.L.D.  is  spoken  of 
as  ten  times  normal  (D.A.N.)10,  etc.  One  c.c.  of  the  normal 
serum  is  said  to  contain  one  "  immunization  unit."  Work- 
ing back  from  quantities  of  serum  of  known  strength  (anti- 
toxic) and  preserved  in  a  dried  state  in  a  vacuum  and  in 
a  dark  cool  place,  the  potency  of  any  toxin  at  hand  can  be 
determined,  and  against  this  latter,  any  newly  prepared 
serum  can  be  standardized.  In  this  way  a  fairly  uniform 
standard  can  be  maintained.  The  usual  antitoxic  serum 
on  the  market  contains  2000  "  immunity  units  "  or  shortly, 
units  in  4  to  5  c.c.  of  serum  and  equal  to  200,000  M.L.D. 
High-potency  sera  are  prepared  so  that  5  c.c.  contain  5000 
units,  and  correspondingly.  The  serum  keeps  very  well 
for  at  least  one  year  in  a  cool  dark  place.  The  durability 
of  the  serum  is  tested  by  keeping  back  some  of  the  bottles, 
and  from  time  to  time  examining  their  activity,  and  if  it  is 
found  to  rapidly  diminish,  all  the  bottles  bearing  the  same 
number  are  recalled. 

OTHER     IMMUNITY     PHENOMENA. 

Pfeiffer's  Phenomenon.  —  In  1894,  Pfeiffer  showed 
that  when  cholera  spirilla  are  injected  into  the  peritoneal 
cavity  of  cholera-immune  guinea-pigs,  the  spirilla  rapidly 
swell  up,  become  granular,  and  often  undergo  complete 
solution.  The  same  result  could  be  observed  in  a  normal 
animal,  if  a  protective  amount  of  cholera-immune  serum 
were  injected  at  the  same  time.  The  constituents  of  the 
blood  serum  which  cause  this  result  are  spoken  of  as 
"  Bacteriolysins."  It  was  soon  shown  that  the  same 
result  followed  the  mixing  of  the  spirilla  and  the  serum  in  a 
test  tube  under  suitable  conditions.  The  same  phenomenon 
was  thereafter  observed  for  other  micro-organisms. 

Agglutination.  —  In  1896,  Grueber  and  Durham, 
investigating  Pfeiffer's  phenomenon,  found  that  when  a 


192         PUBLIC    HEALTH    BACTERIOLOGY 

quantity  of  immune  serum  is  added  to  a  broth  culture  of 
the  respective  bacterium,  flake-like  clumps  sink  to  the 
bottom  of  the  tube,  and  the  supernatant  liquid  becomes 
clear.  Grueber  also  showed  that  the  immune  serum 
would  affect  in  the  same  way,  though  less  powerfully, 
closely  alhed  bacteria.  The  substances  causing  this  are 
called  "  Agglutinins,"  and  were  thought  to  be  the  same  as 
the  "  immune-body M  concerned  in  Pfeiffer's  reaction. 
Both  are  comparatively  thermostabile,  but  the  agglutinins 
cannot  be  reactivated  by  the  subsequent  addition  of  normal 
serum.     They  do  not  act  if  NaCl  is  absent. 

Precipitation. — In  1897,  Kraus  showed  that  precipi- 
tates were  formed  when  filtrates  of  cultures  were  mixed  with 
the  corresponding  immune  serum.  The  "  Precipitins," 
like  the  agglutinins,  are  inactivated  by  heat  (6o°  to  700  C.) 
and  cannot  be  reactivated  (see  Serum  Precipitation, 
below). 

Haemolysis. — Bordet,  in  1898,  showed  that  the  serum 
of  an  animal  which  has  been  repeatedly  injected  with  the 
red  corpuscles  of  another,  acquires  the  power  of  dissolving 
the  red  cells  of  that  other,  and  that  this  power  is  lost  on 
heating  to  550  C.  but  is  regained  by  the  addition  of  serum 
of  a  non-treated  animal.  Other  "  cytotoxins "  (cell- 
destroying  antibodies)  similarly  produced  are  :  leucotoxin, 
nephrotoxin,  spermato toxin,  hepatotoxin,  pancreatoxin, 
suprarenal  toxin,  etc. 

Serum  Precipitation. — Like  haemolysis,  this  subject 
is  closely  allied  to  the  reactions  induced  by  bacteria. 
When  the  serum  of  one  animal  is  injected  repeatedly  into 
another  animal  of  a  different  species,  a  substance  forms  in 
the  first  animal's  serum,  which  causes,  in  a  mixture  of  the 
two  sera,  a  cloudiness  or  precipitate  to  form.  This 
substance  is  called  "  precipitin,"  and  is  specific  for  each 
species,  in  high  dilutions,  as  in  the  case  of  the  other 
reactions.  The  precipitins,  whether  formed  from  bacterial 
or  serum  stimulation  (or  casein  of  milk,  etc.),  are  all 
inactivated  by  heating  to  6o°  to  700  C,  but  can  not  be 
reactivated  by  the  addition  of  normal  serum  or  any  known 
method.  Such  inactivated  serum,  however,  if  mixed 
with  a  certain  amount  of  active  serum,  is  able  to  prevent 


IMMUNITY    AND    ANAPHYLAXIS  193 

the  latter  giving  a  precipitate.  The  precipitins  are  there- 
fore conceived  to  be  built  up  of  two  atom  groups,  one 
thermolabile  and  the  other  thermostabile.  Unlike 
agglutinins,  they  have  not  been,  so  far,  shown  to  exist  in 
normal  serum.  This  reaction  is  used  in  forensic  work, 
to  determine  the  character  of  blood-stains,  whether  human 
or  not. 

Opsonic  Action. — In  1903-4,  Sir  Almroth  Wright 
undertook  a  systematic  study  of  the  phenomenon  of 
phagocytosis,  and  showed  that  phagocytosis  depended 
on  a  substance  present  in  the  serum,  which  acts  on  the 
bacteria  (and  not  on  the  leucocytes),  becomes  fixed  to 
them,  and  makes  them  a  prey  to  the  leucocytes.  To  this 
substance  he  gave  the  name  "  opsonin  "  (feast  preparer). 
This  substance  is  present  in  normal  serum,  but  can  be 
increased  by  immunization.  It  is  destroyed  by  heating 
to  55°  C.  Leucocytes  washed  with  salt  solution  have  no 
phagocytic  action.  On  the  other  hand,  if  the  bacteria  are 
exposed  to  the  action  of  serum,  and  then  washed  free  of  it, 
they  can  be  phagocytozed  by  the  washed  leucocytes.  It  is 
hence  inferred  that  the  opsonin  becomes  bound  to  the 
bacterium.  A  similar  substance  has  been  described  in 
immune  sera  after  heating  (bacteriotropin),  but  it  is 
specific  for  the  corresponding  bacterium,  while  the  opsonin 
in  normal  serum  is  non-specific.  There  are  thus  two 
opsonic  substances ;  that  present  in  normal  serum,  which  is 
thermolabile,  and  that  present  in  immune  sera,  which  is 
thermostabile.  In  opsonic  estimation,  both  factors  are 
at  work  where  the  person  is  being  gradually  immunized 
to  a  particular  bacterium.  The  whole  question  is  still 
complex  and  full  of  difficulties,  and  requires  further 
elucidation. 

Technique  of  Opsonic  Estimation. — The  method  of 
Leishman  is  very  simple.  Take  a  capillary  pipette,  fitted 
with  a  rubber  nipple.  Make  a  mark  on  the  stem  ;  draw  up 
fresh  blood  from  the  finger  to  the  mark.  Then  draw  up 
a  little  air  to  make  an  air-bubble,  which  separates  the 
blood  from  the  bacterial  emulsion  now  drawn  up  to  the 
same  mark.  The  two  fluids  are  then  mixed  by  being 
blown  out  on  a  glass  slide,  and  drawn  back  repeatedly. 
Finally,  the  drop  is  placed  on  the  slide,  covered  with  a 

13 


194         PUBLIC    HEALTH    BACTERIOLOGY 

cover -slip,  and  incubated  at  370  C.  for  15  minutes. 
Thereafter  a  film  is  made  and  stained  by  Leishman's 
method,  and  the  number  of  bacteria  present  in  50  poly- 
morphonuclear leucocytes  is  observed.  This  number 
divided  by  50  gives  the  "  opsonic  index."  Wright's 
method  is  more  elaborate  and  specific.  He  uses  (1) 
Leucocytes  from  the  observer's  blood  repeatedly  washed 
with  saline  solution  (o-85  per  cent)  ;  (2)  Bacterial  emulsion 
in  salt  solution  ;  (3)  Serum  from  the  blood  to  be  tested,  free 
of  clot  and  cells.  These  are  now  mixed,  as  above,  in  equal 
amounts,  in  a  capillary  pipette,  and  the  mixing  is  made 
thorough  by  blowing  out  and  sucking  in,  for  ten  times. 
The  mixture  is  then  drawn  into  the  tube  of  the  pipette, 
the  end  sealed,  the  rubber  nipple  removed,  and  the  tube 
put  into  the  incubator  for  15  minutes.  The  tube  is  then 
removed,  the  end  broken  off,  the  contents  are  again  mixed, 
and  films  made,  dried  or  fixed,  and  stained  as  desired 
(Jenner,  Leishman,  or  Giemsa),  and  bacteria  counted  in 
50  to  100  cells.  A  control  is  done  with  normal  serum,  and 
the  "Opsonic  Index"  taken  is  the  quotient  of  that  got 
with  the  patient's  serum  divided  by  the  index  got  with 
the  normal  serum.  The  latter,  to  minimize  variation, 
may  be  made  by  mixing  the  serum  of  several  healthy 
individuals.  The  number  of  bacteria  per  leucocyte 
(polymorphonuclear),  in  any  one  estimation,  is  also  spoken 
of  as  the  "  Phagocytic  Index,"  and  the  opsonic  index  is 
thus  the  proportion  between  the  phagocytic  index  for  the 
patient's  serum  and  that  for  the  normal  serum.  A 
modification  of  the  method  is  to  take  a  number  of  dilutions 
of  the  patient's  serum  and  of  the  normal  serum  and  to 
estimate  in  all  these  not  the  phagocytic  index,  but  the 
percentage  of  leucocytes  which  act  as  phagocytes,  i.e., 
the  "Percentage  Index,"  and  the  indices  of  the  corres- 
ponding dilutions  can  be  compared.  The  bacterial 
emulsion  used  is  likewise  thinner  than  in  Wright's  method. 
By  the  same  process,  the  dilution  of  the  serum  at  which 
phagocytosis  is  absent  or  very  slight,  can  be  determined. 
This  is  called  the  "  Opsonic  Coefficient  of  Extinction." 
Wright's  Vaccine  Therapy  consists  in  injecting  killed 
bacterial  cells  into  the  infected  individual,  in  order  to 
raise  the  phagocytic  index   to  that  particular  cell.     He 


IMMUNITY    AND    ANAPHYLAXIS  195 

began  with  chronic  staphylococcal  infections,  and  in  such 
cases  good  results  were  obtained.  The  method  has  been 
applied  to  infections  by  tubercle  bacilli,  streptococci, 
gonococci,  pneumococci,  and  other  bacteria.  If  possible, 
a  culture  is  made  from  the  actual  bacteria  causing  the 
infection.  From  the  culture  so  made,  an  emulsion  in 
sterile  salt  solution  is  obtained,  and  the  emulsion  sterilized 
at  the  lowest  possible  temperature  (usually  65  °  C.  for  one 
hour),  and  an  agar  inoculation  made  from  the  presumably 
sterile  emulsion,  and  incubated  for  twenty-four  hours 
to  see  if  really  sterile.  The  bacterial  content  of  the 
emulsion  must  be  estimated,  so  as  to  be  able  to  know  how 
many  are  being  injected,  and  to  inject  a  definite  quantity. 
This  is  done  by  mixing  equal  quantities  of  the  emulsion 
(whole  or  diluted)  and  fresh  blood,  and  making  films,  and 
staining.  The  number  of  bacteria  to  red  cells  is  noted 
over  a  field  made  by  drawing  a  circle  with  a  blue  pencil 
on  the  lens  of  the  eye-piece  of  the  microscope.  The 
number  of  red  cells  in  the  blood  being  known  (say  5  million 
per  c.mm.),  and  the  relative  proportion  of  bacteria  in 
undiluted  emulsion  to  the  red  cells  being,  say,  700  to  400, 
then  as  400  :  700  :  :  5,000,000  :  x  =  8,750,000  bacteria  per 
c.mm.  If  the  emulsion  had  been  diluted,  then  the  result 
would  have  to  be  multiplied  by  the  number  of  the  dilutions. 
It  is  preferred  that  the  content  be  estimated  before 
sterilization,  as  some  of  the  bacteria  may  undergo 
disintegration  during  that  process.  Where  the  blood 
serum  has  an  agglutinating  effect,  the  red  cells  are  separated 
and  mixed  with  salt  solution.  It  is  better  to  render 
motile  bacilli  still  by  having  a  little  formol  in  the  saline 
solution.  From  the  stock  emulsion  thus  standardized 
and  sterilized,  appropriate  doses  are  made  by  dilution 
with  0-5  per  cent  phenol  or  lysol  solution,  and  put  in  glass 
bulbs,  with  capillary  ends  which  are  sealed.  The  ordinary 
dosage  is  to  begin  with  100  million  and  to  repeat,  if  necessary 
using  a  larger  dose,  after  estimating  the  opsonic  index,  and 
only  if  this  is  rising.  Numerous  observations  after  the 
injection  of  such  vaccines  have  shown  that  the  opsonic 
indexi^falls  for  some  time  thereafter  ("  Negative  Phase  ") 
and  then  begins  to  rise,  and  usually  ascends  to  a  higher 
level  than  before.     Another  injection  during  the  negative 


196         PUBLIC    HEALTH    BACTERIOLOGY 

phase  tends  to  accentuate  it,  while  after  the  increasing  or 
"  Positive  Phase  "  has  begun,  an  injection  causes  it  to 
reach  a  still  higher  level.  The  negative  phase  is  usually 
completed  in  twenty-four  hours,  and  the  positive  in  three 
to  four  days.  Wright  recommends  that  the  succeeding 
injection  should  be  given  when  the  positive  phase  has  just 
reached  its  summit.  In  tuberculosis,  Koch's  bacillary 
emulsion  is  used,  and  the  dose  is  minute,  ^qtj-  mgr-> 
gradually  increased  to  TTrVr7  mgr-  In  vaccination  against, 
and  in,  enteric  fever,  a  standardized  strain  of  Bacillus 
typhosus  is  used. 

Leucocyte  Extract. —  The  action  of  the  leucocytes  in 
phagocytosis,  and  of  the  alexines,  which  some  believe  to 
be  derived  from  them,  led  Hiss  to  experiment  with  leuco- 
cytic  extracts.  These  were  obtained  by  the  intrapleural 
injection  of  aleuronat,  which  produced  a  copious  cellular 
exudate  in  24  hours.  The  animal  being  used  (a  rabbit)  is 
killed,  and  the  exudate  removed,  with  every  precaution  to 
ensure  avoidance  of  contamination,  and  the  cells  are 
obtained  by  centrifugalization.  The  deposited  cells  are 
treated  with  sterile  distilled  water,  and  thoroughly  beaten 
with  a  platinum  spatula.  Smears  are  made,  stained  by 
Jenner's  method,  and  examined  for  bacterial  contamination ; 
cultures  are  made  to  detect  the  same  ;  more  sterile  water 
is  added,  and  the  steps  are  repeated  after  incubating  for 
8  hours.  If  no  bacteria  are  found,  the  resulting  fluid  is  put 
into  the  refrigerator  until  used.  Such  extracts  of  exudate 
cells,  on  intraperitoneal  or  subcutaneous  injection,  have 
markedly  modified  the  course  of  infections  in  the  rabbit 
and  guinea-pig,  prolonging  life,  and  in  some  cases  prevent- 
ing a  fatal  issue  from  an  otherwise  lethal  dose.  Beneficial 
effects  have  been  observed  in  man  in  lobar  pneumonia, 
erysipelas,  and  in  staphylococcal  infections.  The  action 
of  the  extract  on  the  bacterial  products  or  toxins  seems 
to  be  a  neutralizing  or  destroying  one.  The  substances 
present  in  these  extracts  have  been  called  "  endolysins," 
and  are  different  from  the  serum  bacteriolysins,  (1)  in 
not  being  inactivated  under  8o°  C.  ;  (2)  when  heated 
above  8o°  C.  they  are  destroyed,  and  cannot  be  reactivated 
by  the  addition  either  of  fresh  serum  or  of  unheated 
leucocyte  extract.     They  are  not  increased  by  immuniza- 


IMMUNITY    AND    ANAPHYLAXIS  197 

tion,  each  leucocyte  probably  having  a  definite  quantity 
within  its  substance. 

Aggressins. — The  great  susceptibility  of  some  species 
of  animals  to  infection  by  certain  bacteria,  while  their 
serum  nevertheless  possessed  marked  bactericidal  power 
against  these  bacteria,  suggested  to  Bail  the  theory  that 
these  bacteria  secrete  definite  substances,  which  protect 
them  against  phagocytosis.  Such  substances  he  called 
"  aggressins,"  and  they  are  therefore  antagonistic  to  the 
opsonins.  They  are  probably  not  produced  in  test  tube, 
or  only  to  a  slight  degree.  He  based  this  theory  on  two 
observations,  namely,  that  sub-lethal  doses  of  bacteria, 
injected  along  with  a  small  quantity  of  "  aggressins"  were 
rapidly  fatal ;  and  that  animals  could  be  immunized  against 
the  corresponding  bacteria  by  the  injection  of  the  aggressins. 
The  aggressins  were  got,  as  detailed  on  page  177,  in  the 
exudate  into  a  serous  cavity  of  an  animal  killed  by  the 
injection  of  a  dose  of  a  particular  bacterium  into  the  serous 
cavity,  and  from  which  exudate  the  bacteria  are  carefully 
removed.  This  theory  has  been  attacked  on  the  ground 
that  the  aggressins  are  merely  the  bacterial  toxins,  probably 
endotoxins,  liberated  in  the  living  body.  The  fact  that 
such  exudates  are  usually  cellular,  along  with  what  has 
been  said  above  of  the  action  of  leucocyte  extract  in  some 
infections,  tends  to  confirm  this  criticism.  In  fact,  it  may 
be  put  this  way  :  When  there  is  a  high  natural  resistance 
to  a  bacterium,  the  alexines  and  opsonins  are  able  to 
overpower  it  in  all  average  infections  and  prepare  it  for 
phagocytosis  and  subsequent  destruction.  On  the  other 
hand,  where  the  natural  resistance  is  low,  these  agents  do 
not  succeed  in  preventing  the  growth  of  the  bacterium, 
which  in  its  growth  elaborates  various  substances,  some 
of  which  reduce  the  resistance  still  further,  and  so  progres- 
sive infection  results.  If  this  infection  is  not  too  severe, 
the  immunizing  apparatus  throughout  the  body,  stimulated 
by  the  diluted  toxins  (extra-  or  infra-cellular)  present  in 
the  blood  stream,  produces  an  excess  of  antibodies  and 
anticells  (phagocytes),  and  in  this  way  may  attain  the 
objective  of  active  immunization.  If  the  infection  is  too 
acute,  paralysis  of  the  immunizing  apparatus  is  the  result. 


198         PUBLIC    HEALTH    BACTERIOLOGY 

THEORIES     OF     IMMUNITY. 

The  rational  explanation  of  all  the  phenomena  of 
immunity  which  are  known,  is  a  task  yet  to  be  accomplished, 
It  is  perhaps  better  to  have  a  working  hypothesis  only, 
as  our  ideas  are  being  continually  enlarged  and  modified. 
The  many  elaborate  and  complex  experiments  which  have 
been  performed  and  repeated  by  many  observers  are 
attended  by  so  many  consenting  circumstances,  of  some 
of  which  we  are  totally  ignorant,  that  it  is  not  surprising 
that  the  inferences  from  the  same  experiment  are  so  varied 
and  even  at  times  so  conflicting.  The  words  of  Pasteur, 
used  in  another  regard,  seem  quite  appropriate  here : 
"  In  experimental  science,  it  is  always  a  mistake  not  to 
doubt,  when  facts  do  not  compel  affirmation.  ...  In  my 
opinion  the  question  is  whole  and  untouched  by  decisive 
proofs." 

Any  theory  must  take  account  of  phagocytosis,  the 
bactericidal  power  of  normal  serum,  the  results  of  immun- 
ization as  seen  in  the  formation  of  antitoxins,  bacterio- 
lysins,  agglutinins,  precipitins,  and  opsonins,  and  any  other 
phenomena  which  emerge  in  the  further  consideration  of 
these.  When  the  theory  is  built  around  the  phagocyte, 
it  is  called  a  "  cellular  "  theory  ;  if  the  body  fluids  are 
taken  as  the  key,  a  "  humoral "  theory.  The  final 
explanation  will  probably  lie  in  a  judicious  blending  of 
these  two  theories. 

Metchnikoff's  Phagocytic  Theory.  —  In  this  theory 
immunization  leads  to  a  more  rapid  and  greater  leuco- 
cytosis  in  response  to  subsequent  infection  by  the  same 
agent.  At  the  seat  of  invasion  there  is  also  emigration  of 
the  microphages  from  the  blood-vessels  into  the  tissues, 
or  if  in  a  serous  cavity,  the  exodus  is  into  the  same,  giving 
a  cellular  exudate.  On  examination  of  these  cells,  many 
of  them  are  found  (in  bacterial  infections)  to  contain 
bacteria  in  their  substance.  These  are  not  simply  dead 
bacteria,  in  process  of  removal,  but  living  and  virulent 
ones.  At  a  later  stage,  the  bacteria  may  be  seen  swollen, 
granular,  and  vacuolated,  and  finally  disintegrated.  On 
the  other  hand,  the  phagocyte  may  not  be  able  to  digest 
the  engulfed  bacteria,  and  may  itself  be  killed ;  and  in  that 


IMMUNITY    AND    ANAPHYLAXIS  199 

case,  the  leucocyte  itself  will  disintegrate.  The  substance 
present  in  normal  blood,  which  is  bactericidal,  Metchnikoff 
calls  "  Cytase,"  and  he  holds  that  it  is  secreted  by  the 
phagocytes.  The  substance  formed  in  immunization  which, 
like  the  cytase,  is  bactericidal,  he  calls  the  "  Fixateur," 
and  also  looks  upon  as  a  derivative  of  the  leucocytes.  He 
believes  that  these  substances  (at  least,  the  cytase)  are  only 
set  free  in  the  blood-stream  by  the  destruction  of  the 
phagocytes.  The  action  of  opsonins  and  leucocyte  extract 
all  tend  to  confirm  the  importance  of  phagocytosis,  and 
the  probability  that  these  cells,  retaining  the  characters  of 
the  amoeba,  retain  also  its  marvellous  adaptability,  which 
is  not  usually  seen  or  expected  of  the  fixed  tissue  cells, 
which  are  so  very  highly  specialized  as  to  function. 
Metchnikoff  therefore  believes  that  for  every  infection 
the  leucocytes  develop  a  power  of  resistance,  which  may  be 
revived  on  any  subsequent  infection,  and  so  protect  to 
a  greater  or  less  degree. 

Ehrlich's  Theory  is  more  complex.  The  discovery 
of  antitoxins  led  to  explanations  of  their  action.  At  first 
they  were  thought  to  destroy  the  toxin,  but  this  simple 
explanation  was  set  aside  by  the  experiments  of  Calmette 
on  snake  poison,  which  is  thermostabile  up  to  ioo°  C.  He 
noted  that  non-toxic  mixtures  of  the  toxin  and  antitoxin 
became  toxic  again  on  heating,  the  inference  being  that 
the  toxin  was  bound  or  inactivated  by  the  antitoxin, 
which  is  destroyed  on  heating  above  6o°  C,  and  so  the 
more  stabile  toxin  is  again  left  free.  Further,  C.  J.  Martin 
and  Cherry  demonstrated  the  close  resemblance  of  the 
union  to  that  of  definite  organic  compounds,  by  an 
experiment  in  which  they  tried  to  pass  toxin-antitoxin 
mixtures  through  a  Chamberland  bougie,  the  pores  of 
which  were  filled  with  gelatin.  In  previous  experiments 
they  found  that  under  50  atmospheres  of  pressure,  toxin 
passed  through  but  antitoxin  did  not.  In  toxin-antitoxin 
mixtures,  if  filtered  at  once,  all  the  toxin  came  through  ; 
but  after  standing  for  variable  periods,  less  came  through 
the  longer  the  time,  until  two  hours  after  mixing,  no  toxin 
passed  through  the  filter  (or  dialyser) .  Then  Ehrlich 
showed,  using  ricin  and  antiricin,  that  definite  quantitative 
proportions  of  the  toxin  and  antitoxin  entered  into  the 


200         PUBLIC    HEALTH    BACTERIOLOGY 

reaction.  The  standardization  of  diphtheria  toxin  and 
antitoxin  was  the  next  step.  Von  Behring  called  a  toxin 
containing  ioo  minimum  lethal  doses  (for  a  250  grm.  guinea- 
pig)  in  1  c.c,  a  "normal  toxin  solution"  (D.T.N^M250), 
and  a  serum  capable  of  neutralizing  it  c.c.  for  c.c,  a 
"  normal  antitoxin  "  or  an  "  antitoxin  unit." 

Ehrlich,  in  working  at  the  subject,  more  exactly 
measured  the  toxin  unit  by  introducing  a  time-limit, 
namely,  that  one  unit  must  kill  the  guinea-pig  in  4  to  5 
days.  He  also  varied  von  Behring's  method  of  testing 
the  antitoxin,  by  first  mixing  the  toxin  and  antitoxin 
outside  the  body,  and  thereafter  injecting  ;  whereas  von 
Behring  injected  them  separately  and  at  different  parts. 
He  prepared  in  this  way  an  antitoxin,  which  he  kept  in  a 
stable  condition  by  drying  in  a  vacuum  and  preserving 
in  the  dark  in  a  dry  atmosphere  and  at  a  low  temperature. 
With  this  antitoxin  he  is  able  to  standardize  new  toxins, 
and  from  them  new  antitoxins.  In  the  course  of  this 
work  he  made  some  discoveries.  In  the  first  place,  while 
the  death  of  a  guinea-pig  in  4  to  5  days  gave  a  fair  measure 
of  1  toxin  unit,  when  100  such  units  were  mixed  with  the 
amount  of  antitoxin  necessary  to  neutralize,  namely, 
1  antitoxin  unit  (which  was  determined  from  previous 
measurement),  and  injected  into  a  guinea-pig,  it  was  not 
easy  to  estimate  whether  there  was  exact  neutralization, 
or  less,  or  more.  If  the  antitoxin  were  markedly  insufficient 
to  neutralize  the  toxin,  then  symptoms  such  as  paralysis, 
etc.,  would  arise  which  would  proclaim  this.  But  the 
conditions  of  the  experiment  were  such  that  no  marked 
signs  could  be  expected.  The  further  test,  therefore, 
was  devised,  namely  to  find  the  amount  of  toxin  which, 
plus  1  unit  of  antitoxin  would  still  be  able  to  kill  a  guinea- 
pig,  on  injection,  in  4  to  5  days.  (When  a  new  serum  is  to 
be  standardized,  the  amount  of  serum  which,  mixed  with 
this  last-mentioned  amount  of  toxin,  just  suffices  to  prevent 
the  death  of  the  guinea-pig  before  4  days,  is  taken  as  one 
unit  of  antitoxin.)  Theoretically  one  might  have  expected 
that,  if  1  toxin  unit  killed  a  250  grm.  guinea-pig  in  4  to  5 
days,  and  1  antitoxin  unit  exactly  neutralized  100  toxin 
units,  the  injection  of  1  antitoxin  unit  mixed  with  101 
toxin  units  would  have  left  1  toxin  unit  free  to  have  killed 


IMMUNITY    AND    ANAPHYLAXIS  201 

the  guinea-pig  in  4  to  5  days.  Piactically  this  was  not 
found  to  be  so,  but  that  a  considerable  excess  of  toxin 
units  is  required  to  kill  the  guinea-pig,  (stated  by  Ehrlich 
to  be  100  toxin  units,  and  by  others  as  intermediate 
amounts).  On  this  basis  Ehrlich  has  built  a  whole  structure 
of  epitoxoids,  toxoids,  prototoxoids,  syntoxoids,  and  toxons, 
designed  to  explain  on  the  laws  of  chemical  equivalence 
and  differences  in  chemical  affinity,  the  apparent  contra- 
diction of  these  results.  It  is  right  to  point  out,  however, 
that  the  basis  is  not  a  chemical  one.  The  death  of  a 
guinea-pig  in  4  to  5  days  cannot  be  compared,  though  it 
follows  on  an  injection  of  toxin,  to  the  appearance,  more 
or  less  immediately,  of  a  precipitate  in  a  liquid  to  which 
some  other  chemical  body  has  been  added.  Also,  the 
relationship  between  the  mixture  of  toxin  and  antitoxin 
which  just  causes  no  symptoms,  and  that  mixture  which 
kills  a  guinea-pig  in  4  to  5  days,  is  an  arbitrary  one,  and 
any  apparent  numerical  relationship  should  be  regarded  as 
fortuitous  until  other  evidence  shows  that  it  is  more  than 
that.  Again,  the  admitted  instability  of  the  toxins,  and 
probably  of  antitoxins,  even  under  every  precaution, 
renders  the  results  equivocal.  In  brief,  Ehrlich's  theory 
is  that  antitoxin  has  a  valency  of  200  for  toxin,  and  that 
some  of  the  bonds  of  antitoxin  can  be  satisfied  by  degenera- 
tion products  of  toxin,  prototoxoids,  deuterotoxoids, 
tritotoxins,  of  alpha  and  beta  varieties,  and  by  toxons, 
which  are  not  derived  from  toxin  but  are  present  in 
the  toxin  fluid  at  first.  ["  Valency  =  200  "  cannot  be 
accepted  in  the  chemical  sense.  It  only  means  that, 
starting  with  an  amount  of  antitoxin  which  neutralized 
100  toxin  units  elsewise  defined,  it  was  found  that  when 
the  conditions  were  changed,  the  antitoxin,  in  some  way 
or  other,  neutralized  200  of  these  same  toxin  units,  or  in 
the  experiment  appeared  to  do  so.  If  the  amount  of 
antitoxin  used  at  first  had  been  the  amount  required  to 
neutralize  1  toxin  unit  (and  von  Behring  advised  the 
larger  quantity  only  for  safety),  the  amount  found  in  the 
second  experiment  would  have  been  2  by  inference.  The 
mixture  of  the  toxin  and  antitoxin  before  injection  may  be 
a  factor  of  moment  in  regard  to  this  change  of  valency.] 
Ehrlich  calls  the  quantity  of  toxin  which  just  neutralizes 


202         PUBLIC    HEALTH    BACTERIOLOGY 

i  antitoxin  unit,  "  limes  zero,"  expressed  as  L0  ;  and 
the  quantity  required  to  neutralize  I  unit  of  antitoxin 
and  yet,  on  injection,  kill  the  guinea-pig  in  4  to  5  days, 
"  limes  death,  expressed  as  L  +.  Then,  according  to  his 
theory,  if  T  represent  1  toxin  unit, 

Lo  =  iooxT  and  L+  =  L0  +  ioixT  ==  20ixT. 

Ehrlich's  side-chain  theory  is  based  on  his  previous 
researches  on  the  oxygen  requirements  of  the  organism, 
linked  up  with  those  on  diphtheria  antitoxin.  Borrowing 
the  language  of  organic  chemistry,  he  likens  the  highly 
complex  albuminous  and  other  molecules  of  animal 
nutrition,  to  those  complex  compounds  of  the  aromatic 
series  which  chemists  have  dissected  into  a  central  group, 
in  which  the  elements  may  be  represented  as  a  hexagon 
or  ring  of  the  benzene  type,  and  the  various  other  parts  as 
side-processes  or  side-chains.  Thus  benzene  has  derivatives 
like  the  following,  the  added  groups  of  which  may  be 
spoken  of  as  side-chains. 

.CH-CEL 

Benzene    CH<  >CH 


CH  -  CH' 


O.CH 


CH  -   C 

Acet.     CH3.CH2.-C/  >C-CO.OCH 

Eugenol  XCH  -  CHX 


Applying  the  same  ideas  to  living  cells,  Ehrlich  believes 
that  these  cells  have  side-chains  which  have  certain  special 
affinities.  .  In  this  way  the  diphtheria  toxin  may  be 
supposed  to  be  bound  to  certain  nerve  cells  ;  and  likewise 
tetanus  toxin.  The  side-chains  he  calls  receptors.  When 
thus  bound  by  a  toxin  molecule,  they  are  supposed  to  be 
useless  to  the  cell  and  are  cast  off  into  the  blood-stream, 
and  the  cell  is  supposed  to  be  stimulated  to  produce  more  ; 
not  only  so,  but  stimulated  to  produce  an  overplus  which 
is  alleged  to  be  then  cast  into  the  blood-stream,  as  the 
cell  would  become  overstocked.  Thus  he  accounts  for 
the  presence  of  antitoxin  free  in  the  blood,  the  free  receptors 
acting  as  antitoxin  to  the  toxin  circulating.  The  toxin, 
which   thus  unites  with  the   antitoxin,   he   conceives   as 


IMMUNITY    AND    ANAPHYLAXIS  203 

having  two  affinities :  one  which  unites  with  the  antitoxin, 
the  haptophore  ;  the  other,  the  toxophore,  by  which  the 
harmful  effects  are  produced.  These  two  affinities  are 
the  affinities  of  two  different  atom-groups,  of  which  the 
toxin  is  supposed  to  be  composed.  In  the  toxoid  bodies,, 
the  toxophore  group  is  altered  or  wanting,  but  they  can 
still  bind  antitoxin,  in  virtue  of  their  haptophore  group. 

For  other  forms  of  immunity,  which  are  more  complex 
than  the  toxin-antitoxin  one,  some  elaboration  is  required. 
In  natural  immunity,  the  blood  contains  a  thermolabile 
substance  which  is  bactericidal.  To  this  substance 
Buchner  gave  the  name  "  Alexine  "  ;  Metchnikoff  spoke  of 
it  as  "  Cytase  "  ;  and  Ehrlich  renamed  it  "  Complement."" 
In  active  immunization,  a  more  thermostabile  substance,, 
bactericidal  in  nature,  appears  in  the  serum,  and  has  been 
variously  called  "Fixateur"  (Metchnikoff),  "Substance 
Sensibilisatrice  "  (Bordet),  "Immune  Body  or  Ambo- 
ceptor "  (Ehrlich).  This  substance  is  found  to  act  only 
in  the  presence  of  complement,  and  hence  Ehrlich' s 
conception  that  it  has  two  combining  affinities  which 
must  be  satisfied  to  produce  bacteriolysis.  The  one 
affinity  binds  it  to  complement,  the  other  to  the  immunizing 
substance  (bacterium  or  red  blood  cell,  leucocytes  and 
other  body  cells,  toxins,  ferments)  ;  called  the  Antibody - 
producer,  or  Antigen.  The  immune  body  he  therefore 
called  an  amboceptor,  or  receptor  with  two  hands,  which 
he  described  as  the  cytophile  haptophore  and  the  comple- 
mentophile  haptophore.  He  also  believes  that  the 
complement  is  composed  of  two  parts,  a  haptophore 
group  and  a  zymophore  group.  According  to  Ehrlich,  the 
complement  is  unable  to  act  directly  on  the  antigen  or 
antibody-producer,  but  only  when  connected  by  the 
immune  body  or  amboceptor.  Bordet,  however,  believes 
that  neither  antigen  nor  immune  body  has  any  affinity 
for  complement,  until  when  they  are  united  they  can 
absorb  the  complement,  but  not  through  the  immune  body. 
The  hypotheses  of  Ehrlich  have  been  used  to  explain 
agglutination,  precipitation,  and  other  phenomena,  with 
sundry  modifications.  It  is  at  present  unnecessary  to  follow 
the  theory  further,  because  in  these  fields  its  explanations 
have  been  most  called  in  question.     This  is  not  to  be 


204         PUBLIC    HEALTH    BACTERIOLOGY 

wondered   at    considering   the  enormously  increased  and 
increasing  complexity  of  the  subject-matter. 

Under  either  theory  of  immunity,  the  immune-body 
produced  in  active  immunization  is  specific,  that  is,  there 
is  a  special  immune-body  produced  for  each  antigen.  On 
the  other  hand,  the  complement,  or  alexine,  or  cytase,  is 
believed  to  be  one  and  the  same,  though  Ehrlich  and  his 
school  have  argued  in  favour  of  specific  complements  for 
specific  amboceptors. 

FURTHER    IMMUNITY    PHENOMENA. 

These  can  be  more  easily  followed  after  the  terms  used 
in  the  theories  have  been  acquired. 

i.  Filtration  of  Serum. —  Muir  and  Browning  found 
that  on  filtering  serum  through  a  Chamberland  bougie,  the 
immune-body  passed  through,  and  the  complement  did  not. 

2.  Fixation  of  the  Complement. — Bordet  and  Gengou 
planned  an  experiment,  called  the  M  Bordet  and  Gengou 
Reaction,"  to  demonstrate  the  presence  in  a  given  serum 
of  a  specific  immune-body,  even  in  very  small  quantities. 
To  this  reaction  the  term  "  Fixation  of  the  Comple- 
ment "  is  now  commonly  applied,  and  has  its  best  known 
practical  use  in  the  "  Wassermann  Reaction."  They 
performed  two  parallel  experiments,  (i)  and  (2),  in  which 
they  used  the  following  mixtures  : — 

(1).  Heated  immune  plague  serum  -f  plague  bacilli 
emulsion  -f  fresh  normal  serum. 

(2).  Heated  normal  serum  +  plague  bacilli  emulsion  -f- 
fresh  normal  serum. 

Set  aside  for  five  hours  at  blood  heat.  Then  added  to 
each  mixture : 

(a)  geated  hemolytic  serum  +  washed  red  blood  cells. 

Obs*e?v^  results  :  Mixture  (1)  shows  no  haemolysis  ; 
mixture  (2)  shows  haemolysis. 

The  explanation  of  this  phenomenon  is  after  this 
manner  :  Looking  at  (a)  we  see  that  the  mixture  there 
requires  the  addition  of  complement  to  produce  haemolysis, 
since  the  complement  in  the  serum  has  been  destroyed  by 
heat.  Therefore,  when  mixture  (2)  produces  haemolysis, 
we  infer  that  it  must  have  supplied  complement ;    and 


IMMUNITY    AND    ANAPHYLAXIS  205 

similarly  that  mixture  (i),  since  it  did  not  produce  haemo- 
lysis, could  not  have  supplied  any  complement.  We  have 
therefore  to  examine  mixtures  (i)  and  (2)  carefully  to 
determine  the  cause  of  their  different  actions.  To  do  this,, 
they  may  be  rewritten  thus  : — 

(1).  Specific  immune-booly  -f-  specific  antibody-producer 
-f-  complement. 

(2) .  Non  -  specific  immune  -  body  -f-  specific  antibody- 
producer  -f-  complement. 

They  differ  only  in  their  immune-bodies  (normal  serum 
is  believed  to  contain  non-specific  immune- bodies) .  It  is 
therefore  inferred  that  in  (1),  since  no  complement  is  left 
free,  it  has  become  bound  by  the  joint  action  of  the  specific 
immune-body  and  the  corresponding  antibody-producer. 
In  (2),  since  the  immune-body  and  the  antibody-producer 
do  not  correspond,  no  binding  or  fixation  of  the  complement 
occurs,  and  so  haemolysis  takes  place  on  adding  (a).  It 
should  be  noted  here,  that  the  quantity  of  complement 
used  is  determined  by  previous  experiment,  as  the  presence 
of  an  excess  over  the  quantity  which  can  be  bound  by  the 
amounts  of  immune-body  and  antibody-producer  would 
lead  to  haemolysis,  even  in  (1). 

This  reaction  is  capable  of  many  applications  in  the 
determination  of  specific  immune-bodies  in  a  serum  and  of 
specific  antihoffo-prorhicers  (antigens)  in  a  serum.  We 
shall  here  describe  briefly  the  so-called  "  Wassermann 
test  "  for  the  diagnosis  of  syphilis,  by  determining  the 
presence  in  the  patient's  blood  of  an  immune  body,  capable 
when  mixed  with  syphilitic  antibody-producer  (antigen) 
of  causing  fixation  of  complement. 

Wassermann  Test. — Requisites  : 

(1)  Specific  antibody-producer,  referred  to  usually  as 
the  antigen. 

(2)  Red  blood  cells  of  a  sheep,  washed  free  of  com- 
plement. 

(3)  Serum  of  a  rabbit's  blood,  haemolytic  to  sheep's  red 
cells,  heated  before  use,  to  destroy  its  complement. 

(4)  Fresh  guinea-pig's  serum,  to  supply  complement. 

(5)  Serum  from  the  patient,  heated  to  560  C,  which 
serum  is  to  be  tested  for  the  presence  of  specific  immune- 
body  or  antibody. 


206         PUBLIC    HEALTH    BACTERIOLOGY 

i.  The  antigen  at  first  used  was  a  salt  solution  extract 
of  the  liver  of  a  syphilitic  foetus.  Alcoholic  extracts  have 
also  been  used,  and  it  has  been  discovered  that  the 
-extracts  of  normal  liver  and  spleen,  and  even  a  I  per  cent 
emulsion  of  lecithin,  will  act  as  antigen.  (This  shows  how 
little  we  must  know  of  possible  fallacies,  in  tests  like  this, 
with  highly  complex  bodies  like  liver  extract.  It  does 
not  impugn  its  specificity  for  pure  antigens,  though  it 
suggests  the  possibility  of  two  different  substances,  with 
similar  affinities  for  the  immune-body  produced  by  one  of 
them.  Of  course  the  probability  of  this  other  substance 
being  present,  under  the  conditions  of  the  experiment,  is 
small,  but  must  never  be  lost  sight  of.)  The  quantity  of 
antigen  to  be  used  has  to  be  carefully  predetermined,  as 
large  excess  of  antigen  has  been  found  to  cause  binding  of 
the  complement,  even  in  the  absence  of  the  immune-body. 
A  series  of  trials  of  varying  quantities  of  antigen  with  the 
same  amount  of  complement  in  each  case,  shows  the 
largest  quantity  of  antigen  which  can  be  used  without 
exerting  this  action.  This  amount  of  antigen  is  arranged, 
by  proper  dilution,  to  be  present  in  i  c.c. 

2.  Washed  Red  Blood  Cells. — Some  sheep's  blood  is 
gathered  under  aseptic  precautions  into  a  small  sterile 
flask,  containing  sterile  solution  of  sodium  citrate  (0-5  per 
cent)  and  sodium  chloride  (0-85  per  cent).  The  corpuscles 
are  separated  by  cehtrifugalizing,  and  washed  repeatedly 
in  the  same  way  with  sterile  salt  solution,  to  get  rid  of 
serum-complement  and  serum-precipitins.  They  are  then 
brought  down  to  a  5  per  cent  emulsion  in  salt  solution,  by 
mixing  them  with  19  times  their  bulk  of  the  same. 

3.  Hemolytic  Serum  for  Sheep's  Cells. — This  is  obtained 
by  injecting  a  rabbit  with  washed  red  blood  cells  of  a  sheep, 
obtained  as  above.  Of  the  5  per  cent  emulsion,  three  or 
four  injections  are  given  at  intervals  of  5  to  6  days  ;  the 
first  injection  of  5  c.c,  the  second  of  10  c.c,  the  third  of 
15  c.c,  and  the  fourth  of  20  c.c.  ;  intravenously  or  intra- 
peritoneally.  Ten  days  later  than  the  final  injection  the 
serum  is  obtained  by  drawing  blood  from  the  carotid 
artery,  allowing  it  to  clot,  and  pipetting  off  the  serum. 
A  high-potency  serum  is  desirable  so  that  a  small  quantity 
only  may  be  required.     This  obviates  or  reduces  the  risk 


IMMUNITY    AND    ANAPHYLAXIS  207 

of  precipitates,  due  to  precipitins  in  the  rabbit's  serum  for 
sheep's  serum  (from  insufficient  washing  of  the  injected 
corpuscles),  acting  on  any  sheep's  serum  present,  from 
insufficient  washing  of  the  corpuscles  used  in  the  test.  The 
serum  is  inactivated  by  heating  to  560  C.  The  quantity 
of  haemolytic  serum  to  be  used  must  be  accurately  deter- 
mined,' in  relation  to  a  definite  amount  of  complement. 
This  is  the  more  necessary  since  it  has  been  shown  that, 
(contrary  to  Ehrlich's  commonly  accepted  conception 
that  immune -body  and  complement  react  in  definite 
combining  proportions),  the  haemolytic  immune-body  and 
a  complement  react  in  inverse  proportions  ;  that  is,  the 
more  immune-body,  the  less  complement  is  required.  The 
bearing  of  this  on  the  test  is  that  a  very  small  quantity  of 
complement  left  over  by  the  combination  of  syphilitic- 
immune-body  and  antigen  might  suffice,  in  presence  of 
large  excess  of  h  hemolytic  immune-body,  to  cause  haemo- 
lysis ;  thus  giving  a  negative  result  in  a  positive  case. 
The  quantity  is  determined  by  putting  up  a  number  of 
mixtures,  each  containing  o-i  c.c.  of  fresh  guinea-pig 
serum  to  give  complement,  and  1  c.c.  of  the  5  per  cent 
emulsion  of  sheep's  corpuscles.  To  each  mixture 
inactivated  haemolytic  serum  is  added,  in  smaller  and 
smaller  quantities.  The  smallest  quantity  which  gives 
complete  haemolysis  is  taken  as  the  unit  amount  to  be  used 
in  the  test.  The  haemolytic  unit  may  therefore  be  defined 
as  "  the  smallest  quantity  of  inactivated  haemolytic 
serum  which,  in  the  presence  of  a  stated  amount  of  com- 
plement, is  able  to  cause  complete  haemolysis  in  1  c.c.  of 
a  5  per  cent  emulsion  of  washed  red  blood  cells." 

4.  The  Complement. — This  is  obtained  by  drawing  blood 
from  a  guinea-pig,  allowing  it  to  clot,  and  pipetting  off  the 
serum,  or  separating  by  the  centrifuge.  Such  serum, 
kept  at  a  low  temperature,  preserves  its  complement  in 
a  fairly  constant  amount  for  three  days. 

5.  The  Serum  to  be  tested  is  got  from  the  patient  under 
aseptic  precautions,  from  the  median  basilic  vein,  from 
the  finger,  or  from  the  ear.  Three  to  four  c.c.  are  with- 
drawn, as  1  c.c.  of  clear  serum  is  required  to  go  over 
the  tests  and  controls.  It  is  inactivated  by  heating  to 
560  C.  in  a  water-bath  for  20  minutes.     Noguchi  advises 


208         PUBLIC    HEALTH    BACTERIOLOGY 

540  C.     Serum   can  also  be  obtained  from  cerebrospinal 
fluid,  etc. 
Process. — 

(a).  In  a  test  tube,  put  the  following  : — 

o*i  c.c.  of  complement  (fresh  guinea-pig  serum). 

0*2  c.c.  of  inactivated  test  serum  (from  patient). 

ro  c.c.  of  standardized  antigen  (liver  extract,  etc.). 

Add  salt  solution  to  make  up  bulk  to  3  c.c.  ; 

shake   thoroughly,    and   place   for    1    hour  in 

incubator  at  37*5°  C. 

(b).  Now  add, 

1*0  c.c.  of  5  per  cent  emulsion  of  sheep's  red  cells. 
2#o   units   of   haemolytic   serum    (determined   as 

above) . 
Shake  thoroughly,  and  incubate  again  at  37-5°  C. 
for  1  to  2  hours. 
(c).  Observe  result. 

(1).  No   hcemolysis.      Immune-body    or    anti- 
body is  present  ;  test  positive. 
(2).  Complete    hemolysis.      Immune  -  body   or 
antibody  is  absent ;    test  negative. 
Several  controls  should  be  put  up  at  the  same  time  to 
preclude   error.     Such  are :    a   test  with    known    normal 
serum  ;   tests  with  antigen  and  complement  alone  to  see 
that  antigen  is  not  fixing  complement  ;   test  of  haemolytic 
serum  and  cells  and  complement  with  and  without  antigen 
to  see  that  haemolysis  is  actually  possible,  and  tests  with 
known    syphilitic   serum  with   and  without    antigen.     In 
all  these  controls,  except  that  with  known  syphilitic  serum 
with  antigen,  complete  haemolysis  should  occur. 

Noguchi  has  modified  the  test  by  using  human  red  cells 
and  an  anti-human  haemolytic  serum.  This  simplifies  the 
test  in  that  the  patient's  red  cells  may  be  used  in  testing 
his  own  serum,  and  in  that  no  antibody  for  his  red  cells 
exists  in  his  own  serum  (human  serum  at  times  contains  an 
antibody  to  sheep's  cells).  Anti-human  haemolytic  serum  is 
prepared  and  standardized  in  a  similar  way  to  that 
described  as  used  in  preparing  anti-sheep  haemolytic  serum. 
Human  serum,  when  used  to  provide  complement,  is  said 
to  vary  more  in  its  content  than  guinea-pig  serum,  to  absorb 
10  times  as  much  immune-body,  and  to  be  less  sensitizing. 


IMMUNITY    AND    ANAPHYLAXIS  209 

Fleming's  modification  is  to  use  the  hemolytic  immune- 
body  in  human  serum  for  sheep's  corpuscles,  and  thus  he 
can  use  the  complement  in  the  patient's  blood.  This 
simplifies  the  process. 

D'Este  Emery  uses  human  red  cells,  sensitized  with 
heated  immune  serum  from  a  rabbit  which  has  been 
injected  with  human  cells.  ,  He  advises  only  5  minutes' 
incubation  in  a  water-bath  at  380  C,  which  very  appre- 
ciably shortens  the  time  taken    for  the  test. 

Browning,  Cruickshank,  and  McKenzie  describe  "  a 
method  of  carrying  out  the  reaction  which  is  very  reliable 
in  practice,  and  depends  on  the  fact  that  the  amount  of 
complement  absorbed  by  a  mixture  of  serum  and  lecithin 
is  increased  on  the  addition  of  cholesterin,  if  the  serum  is 
syphilitic  ;  but  not,  if  the  serum  is  normal.  Accordingly, 
two  series  of  tests  are  carried  out  simultaneously — in  the 
one,  complement  is  added  to  the  mixture  of  serum  and 
lecithin ;  in  the  other,  to  the  mixture  of  serum,  lecithin, 
and  cholesterin.  If  more  complement  is  absorbed  in 
the  second  series  than  in  the  first,  then  the  reaction  is 
positive." 

Value  of  the  Test. — Like  the  agglutination  or  Widal 
test  for  enteric  fever,  the  fixation  of  complement  or 
Wassermann  test  for  syphilis  has  its  limitations. 

It  is  almost  always  negative  in  healthy  subjects,  but 
quite  often  gives  a  positive  reaction  in  those  suffering  from 
leprosy,  scarlet  fever,  jaundice,  yaws,  sleeping-sickness, 
and  the  acute  stage  of  malaria. 

In  the  primary  stage  of  syphilis,  a  negative  result  is 
usually  got  in  the  first  fortnight.  Thereafter  one  may 
expect  a  positive  resjilt  in  50  per  cent  of  the  cases,  and 
its  absence  has  some  weight. 

In  the  secondary  stage,  in  the  presence  of  a  suspected 
rash,  a  positive  reaction  is  got  in  50  to  70  per  cent  of 
the  cases. 

In  the  tertiary  stage,  with  progressive  lesions  present, 
the  absence  of  the  reaction  is  almost  conclusive  proof  that 
another  diagnosis  than  syphilis  must  be  sought. 

In  later  stages,  a  positive  reaction  shows  that  the  patient 
is  not  cured.  Does  a  negative  reaction  show  that  he  is. 
cured  ?     If  a  positive  reaction  was  got  previously,  then  it 

14 


210         PUBLIC    HEALTH    BACTERIOLOGY 

is  likely,  always  excluding  treatment  with  mercury,  which 
inhibits  the  reaction. 

If,  at  any  stage,  the  patient  is  under  treatment  with 
mercury,  a  negative  reaction  has  no  value  for  diagnosis. 
Stop  treatment  for  one  to  two  months,  and  test  again. 
Salvarsan  or  "  606  "  also  inhibits  the  test. 

In  a  few  cases  of  syphilis,  no  reaction  is  given  at  any 
time. 

In  congenital  syphilis,  the  reaction  may  persist  through- 
out life,  and  be  present  even  where  there  are  no  evidences 
of  active  pathological  processes.  The  examination  of 
the  blood  of  the  mother  will  usually  give  corroboration, 
and  that  of  the  father  may,  but  not  necessarily. 

In  tabes  dorsalis,  general  paralysis,  and  aneurysm,  a 
large  number  of  positive  results  have  been  obtained ;  about 
60  per  cent  in  tabes,  and  99  per  cent  in  general  paralysis. 
In  the  latter  it  is  got  in  the  cerebrospinal  fluid. 

Porges- Meier  Reaction. — Equal  parts  of  clear  blood 
serum  and  a  1  per  cent  emulsion  of  lecithin  or  other  lipoid 
substance  in  carbolized  salt  solution,  are  mixed  and  allowed 
to  stand  at  room  temperature  for  5  hours.  Normal  serum 
causes  no  precipitate,  but  syphilitic  serum  does.  This  test 
is  not  nearly  so  delicate  macroscopically  as  Wassermann's, 
and  hence,  while  much  more  simple,  is  not  so  readily 
interpreted  by  the  naked  eye.  Jacobsthal  has  studied  it 
microscopically,  by  the  aid  of  the*  ultra-microscope.  He 
found  that  with  normal  serum,  the  particles  of  the  lecithin 
emulsion  appeared  as  isolated  brilliant  points,  showing 
active  Brownian  movements.  With  a  syphilitic  serum, 
these  brilliant  points  were  seen  to  run  together  to  form  a 
large  and  brilliant  mass.  Intermediate  reactions  were 
noted,  in  which  partial  agglomeration  occurred  into  small 
brilliant  masses  or  brilliant  chains.  These  phenomena 
were  tested  against  the  Wassermann  reaction,  and  were 
found  to  run  parallel  to  it. 

Determination  of  an  Antigen  by  Complement- 
Fixation. — The  same  principles  apply,  but  in  this  case 
the  immune-body  present  in  the  serum  is  known,  and  a 
serum  is  tested  which  is  supposed  to  contain  the  corres- 
ponding antigen      It  has  been  applied  to  test  blood  for 


IMMUNITY    AND    ANAPHYLAXIS  211 

the   antigen   of   B.   typhosus,   using  highly  potent   anti- 
typhoid serum  obtained  from  an  immunized  rabbit. 

Deviation  of  the  Complement. — Neisser  and  Wechs- 
berg,  experimenting  with  mixtures  of  specific  inactivated 
immune  sera,  specific  antigen  (bacteria)  and  complement, 
found  that,  beginning  with  the  complement  in  excess, 
more  and  more  bacteria  were  destroyed  as  the  amount 
of  immune -body  was  increased,  up  to  a  maximum. 
Beyond  this,  increase  of  the  amount  of  immune-body 
lessened  bacteriolysis,  and  finally  in  great  excess,  seemed 
to  stop  it  entirely.  To  this  phenomenon  they  gave  the 
name  of  "  deviation  of  the  complement,"  in  accordance  with 
their  theory  that  free  immune-body  has  a  greater  affinity 
for  antigen  than  immune-body  joined  to  complement ; 
and  so  in  great  excess  of  immune-body,  the  immune-body 
appropriates  all  the  bacterial  receptors,  to  the  exclusion 
of  the  immune-body  joined  to  complement ;  hence  the 
cessation  of  bacteriolysis.  This  explanation  cannot  now 
be  accepted,  and  so  the  term  "  deviation  of  the  comple- 
ment "  is  unfortunate  and  should  be  dropped.  It  is 
possible  that  the  explanation  of  the  phenomenon  should 
be  sought  on  physical  lines,  the  excessive  dilution  of  the 
mixture  with  serum  containing  immune-body  reducing 
the  chances  of  bacteriolysis  by  the  complement  in  the  same 
time-limit  ;  and  it  is  possible  the  complement  may  be 
inhibited  by  other  constituents  of  the  serum  added. 

Heterolysins,  Isolysins,  Autolysins. — A  haemolysin 
produced  in  the  blood  of  one  animal  by  the  injection 
of  the  red  cells  of  another  species,  is  called  a  "  hetero- 
lysin."  When  the  hemolysin  is  produced  by  injection 
of  red  cells  of  a  member  of  its  own  species,  it  is  called  an 
"  isolysin."  Both  of  these  have  been  produced.  The 
production  of  a  haemolysin  in  an  animal  by  the  injection 
of  its  own  red  cells,  has  not  been  accomplished.  If  such 
a  haemolysin  were  produced,  it  would  be  called  an  "  auto- 
lysis"  The  injection  of  isolysins  produces  "  anti- 
isolysins,"  which  like  the  heterolysins  and  isolysins  are 
specific.  The  search  for  autolysins  is  clinically  significant, 
as  a  possible  theory  for  paroxysmal  hemoglobinuria. 


212         PUBLIC    HEALTH    BACTERIOLOGY 

ANAPHYLAXIS. 

I.  General  Principles. — 

As  already  defined,  anaphylaxis  is  a  supersensitiveness 
induced  in  man  and  animals  by  the  injection  of  certain 
substances,  mostly,  so  far  as  is  known,  of  an  albuminous 
nature.  The  ideas  on  this  subject  have  grown  around 
anomalous  phenomena  following  on  the  injection  of  diph- 
theria toxin  and  antitoxin.  Thus  v.  Behring  and  others 
noted  that  occasionally  animals  highly  immune  to  the 
toxin  showed  excessive  susceptibility  to  small  doses  of 
the  toxin.  Very  soon  after  the  introduction  of  the  serum 
treatment  of  diphtheria  in  1894,  certain  symptoms  were 
observed  to  follow  the  injection  of  the  serum,  which,  at 
first  ascribed  to  the  antitoxin  contained  in  it,  were  finally 
found  to.  be  due  to  the  horse  serum  which  carried  the 
antitoxin.  These  symptoms  were  hence  called  "  Serum 
Sickness  "  or  "  Serum  Disease  "  and  were  mostly  a  rash 
of  an  erythematous  nature,  and  fever.  These  come  on  in 
most  cases  about  the  ninth  day,  but  vary  from  the  third 
to  the  nineteenth.  Fever  is  not  always  present,  and  the 
rash  is  quite  often  urticarial,  and  occasionally  scarlatini- 
form  or  morbilliform,  is  local  to  the  point  of  injection  or 
becomes  general,  is  fugitive  or  persistent.  Articular 
pains  may  accompany  the  rash,  and  may  be  severe.  The 
frequency  of  these  symptoms  varies ;  from  30  to  55 
per  cent  of  the  cases  treated  with  serum  are  stated  to 
show  them  in  some  degree  or  other.  Some  oedema  is 
also  noted  by  Currie,  as  accompanying  the  rash.  The 
similarity  of  these  symptoms  to  those  of  the  specific 
infective  diseases  is  striking,  and  led  to  the  name  "  serum 
disease,"  which  has  therefore  an  incubation  period  (after 
subcutaneous  injection)  averaging  nine  days,  and  a 
duration  of  two  days  on  the  average.  That  these 
symptoms  were  due  to  the  horse  serum,  and  not  to  the 
antitoxin,  has  been  proved  by  such  symptoms  following 
the  injection  of  the  normal  serum  of  the  horse.  Another 
group  of  symptoms  which  on  rare  occasions  followed  the 
use  of  serum,  was  not  at  once  recognized  as  due  to  it,  but 
by  the  accumulation  of  cases  and  certain  special  features 
connected  with  them,  the  causal  action  of  the  serum  was 


IMMUNITY    AND    ANAPHYLAXIS  213 

obviously  suggested,  and  experiments  on  animals  proved 
the  truth  of  the  inference.  These  symptoms  differed 
from  those  of  serum  disease  in  that  they  came  on  immedi- 
ately or  within  an  hour  after  the  injection,  were  very 
severe,  and  in  some-  cases  ended  fatally.  The  earliest 
recorded  case  is  that  of  the  son  of  Professor  Langerhans, 
who  was  given  a  prophylactic  dose  of  serum,  took  ill  at 
once,  and  died  shortly  afterwards.  The  next  recorded 
cases  were  three  communicated  by  Goodall  to  the  Anti- 
toxin Committee  of  the  Clinical  Society  (of  which 
Committee  he  was  a  member).  The  first  of  these  was 
that  of  a  girl,  aged  4  years,  admitted  to  hospital  suffering 
from  diphtheria.  She  was  given  4000  units  on  October 
24th,  1897,  and  the  same  amount  on  October  25th  and 
26th.  On  Nov.  3rd  there  was  a  slight  urticarial  rash.  On 
Nov.  30th  she  had  a  well-marked  relapse  of  diphtheria,  and 
was  in j  ect ed  with  4000  units  of  antitoxin .  ' '  Within  twenty 
minutes  of  the  injection,  she  was  seized  with  shiverings, 
quickly  followed  by  two  convulsions.  Seen  a  few  minutes 
later  by  the  assistant  medical  officer,  the  convulsions  had' 
ceased,  but  the  child  was  in  a  drowsy  state,  and  the 
temperature  had  risen  to  1050  F.  .  .  .  There  was  no 
rash.  .  .  .  During  the  night  the  child  vomited  several 
times  ;  about  6  a.m.  on  Dec.  1st  a  rash  was  noticed,  and 
was  a  multiform  erythema.  It  persisted  till  Dec.  5th,  and 
while  it  was  present  the  temperature  remained  up  and 
the  pulse  was  very  rapid.  On  Dec.  5th  and  10th  there 
were  twitchings  of  the  mouth,  and  throughout  the  child  was 
drowsy  and  apathetic,  and  had  a  bad  colour.  She  slowly 
recovered  and  left  the  hospital  well  on  Feb.  3rd."  Similar 
cases  could  be  multiplied,  and  some  given  in  which  the 
immediate  reaction  is  limited  to  a  local  or  general  rash. 

Such  cases  giving  an  immediate  reaction  fall  into  two 
groups  :  (1)  Where  the  serum  has  not  previously  been 
injected  ;  and  (2)  Where  a  dose  of  horse  serum  has  been 
administered  on  a  previous  occasion  (excluding  doses 
given  within  the  incubation  period  of  the  serum  disease). 

1.  In  the  first  group  numerous  fatalities  have  been 
recorded.  In  most  of  these,  a  history  of  asthma,  or  some 
similar  condition,  has  been  noted,  and  this  is  very 
important,  since  the  subcutaneous  injection  of  diphtheria 


214         PUBLIC    HEALTH    BACTERIOLOGY 

antitoxin  has  been  recommended  as  a  cure  for  asthma. 
A  record  of  a  fatal  result  in  a  man  of  52  years,  who  died 
in  tonic  spasm  ten  minutes  after  receiving  the  serum, 
elicited  particulars  of  16  similar  cases,  and  in  the 
"  Therapeutic  Gazette "  for  March  15th,  1909,  a  short 
account  is  given  of  these,  with  14  others,  making  30  in  all, 
in  which  alarming  symptoms  followed  shortly  after  the 
injection,  and  ended  fatally  in  16  cases.  In  22  of  the  30 
cases  there  was  a  history  of  asthma  or  some  similar 
affection.     (Goodall  in  Public  Health,  January,  191 1). 

2.  Where  there  is  a  record  of  a  previous  administration 
of  serum,  while  the  immediate  symptoms  may  be  very 
alarming  and  dangerous,  so  far  no  fatality  has  been 
reported.  The  use  of  serum  day  after  day  in  a  severe  case 
of  diphtheria  is  not  followed  by  these  manifestations, 
unless  a  period  of  at  least  ten  days  separates  the  first  dose 
from  that  causing  the  symptoms.  It  is  this  feature  which 
suggested  that  the  symptoms  were  due  to  supersensitive- 
ness  to  serum,  following  on  an  attack  of  serum  disease. 
The  condition,  beginning  ten  days  after  the  first  injection  of 
serum,  has  been  present  in  persons  after  five  years,  and  may 
yet  be  found  at  a  longer  period.  Experimental  work  on 
the  subject  has  shown  that  rabbits  injected  with  horse 
serum  are  very  sensitive  to  a  subsequent  dose,  show  severe 
symptoms,  and  often  die.  This  has  been  called  the 
"  Phenomenon  of  Arthus."  The  "  Phenomenon  of 
Theobald  Smith  "  is  that  guinea-pigs  used  to  standardize 
antitoxin,  when  injected  with  a  toxin-antitoxin  mixture 
were  always  killed  on  the  subsequent  injection,  after  ten 
days,  of  normal  horse  serum.  Otto,  Rosenau,  and  Anderson, 
working  independently,  showed  conclusively  that  the  action 
of  the  serum  was  without  relation  to  its  antitoxin  content  ; 
that  sensitation  of  the  guinea-pig  was  most  marked  after 
10  days  ;  that  very  small  doses  were  efficient  (o-ooi  c.c.  or 
less)  ;  that  the  condition  was  transmissible  from  mother  to 
offspring  ;  that  it  was  specific  for  the  particular  serum 
used  ;  that  it  was  not  a  hemolytic  nor  precipitin  action  ; 
that  the  condition  could  be  conferred  on  another  animal  by 
injecting  it  with  the  serum  of  a  sensitized  animal ;  that  a 
considerable  dose  of  serum  (5  c.c.)  is  required  for  the 
second  injection  ;   and  that  the  symptoms  are  prompter  in 


IMMUNITY    AND    ANAPHYLAXIS  215 

appearing  if  the  second  injection  is  made  intraperitoneally, 
cardially,  or  cerebral  ly,  than  if  given  subcutaneously. 
Sensitized  animals  which  recover  from  the  second  injec- 
tion are  thereafter  immune  (Antianaphylaxis).  This  im- 
munity may  also  be  purchased  by  the  injection  of  large 
quantities  of  the  sensitizing  serum  towards  the  middle  or 
end  of  the  incubation  period,  but  the  duration  of  this 
immunity  is  believed  by  Otto  to  be  short. 

In  a  number  of  cases  where  a  second  injection  is 
given  after  an  interval,  no  immediate  reaction  (within 
24  hours)  follows,  but  an  "Accelerated  Reaction,"  that 
is,  the  local  and  general  symptoms  of  serum  disease  are 
noted  earlier  than  in  the  previous  attack,  or  than  is  the 
average  where  no  record  exists. 

The  simplest  explanation  of  the  phenomena  of  anaphy- 
laxis is  that  of  Wolff-Eisner,  who  holds  that  all  proteid 
substances  contain  a  toxic  part,  which  does  not  produce 
an  antibody  when  injected  into  animals.  On  the  first 
injection  a  lysin  is  formed  which  breaks  up  the  proteid, 
liberating  the  toxic  part.  A  second  injection  results  in 
the  rapid  liberation  of  the  toxic  part  by  the  action  of  the 
already-present  lysin,  and  hence  the  toxaemia.  The 
profound  affection  of  the  nervous  system,  the  general 
vaso-dilatation,  and  the  more  rapid  action  on  intracranial 
injection,  all  suggest  some  substance  which  acts  as  a  toxin 
on  the  nerve  tissues.  In  this  connection  the  use  of  serum 
for  the  cure  of  asthma  is  interesting,  because  it  could  be 
explained  (if  the  patient  survive)  as  diminished  nerve- 
irritability  to  proteid. 

II.  Anaphylaxis  to  White-of-Egg. — 

Besredka  and  Bronfenbrenner  in  their  most  recent 
memoire  (Ann.  de  Vlnstitut  Pasteur,  Mai,  191 1),  have 
studied  very  carefully  the  anaphylaxis  produced  by  the 
injection  subcutaneously  of  white-of-egg,  both  raw  and 
heated  to  ioo°  C.  They  produced  active  anaphylaxis  by 
the  injection  of  0-5  c.c.  of  white-of-egg,  diluted  with  an  equal 
quantity  of  normal  saline  solution.  The  state  of 
anaphylaxis  appeared  in  16  to  20  days  ;  with  a  smaller 
dose  (yj-g-  c.c),  it  appeared  in  12  days.  The  injection 
of  white-of-egg  heated  to  ioo°  C.  into  other  guinea-pigs, 


216         PUBLIC    HEALTH    BACTERIOLOGY 

produced  a  state  of  milder  anaphylaxis  than  the  raw  white- 
of-egg.  They  hence  conclude  that  the  constituent  of  the 
white-of-egg  which  causes  sensitation,  while  attenuated  by 
heat,  is  thermostabile.  This  active  state  of  anaphylaxis 
lasts  for  several  months. 

Passive  anaphylaxis  was  produced  within  24  hours  after 
the  injection  into  a  guinea-pig  of  the  serum  of  a  guinea-pig 
which  had  been  previously  treated  with  white-of-egg.  The 
injection  thereafter  into  the  jugular  vein  of  the  sensitized 
guinea-pig  of  -g^  to  T^  c.c.  of  white-of-egg,  produced 
the  classical  symptoms  of  anaphylaxis,  with  death  in  1 
to  3  minutes.  On  the  other  hand,  when  the  serum  and  the 
white-of-egg  were  injected  at  the  same  time,  one  guinea-pig 
showed  a  marked  dyspnoea  and  some  malaise  immediately 
after  the  injection,  but  quickly  recovered  ;  another  showed 
slight  respiratory  distress  ;  and  a  third  showed  no  reaction. 
In  all  three  the  injection  was  into  the  jugular  vein  ;  in 
the  first  two  it  consisted  of  2  c.c.  of  anaphylactic  serum 
mixed  with  T±-$  c.c.  of  white-of-egg  ;  in  the  third  guinea- 
pig,  it  consisted  of  1-5  c.c.  of  serum  mixed  with  1  c.c.  of 
10  per  cent  solution  of  white-of-egg,  equal  to  -^  c.c.  of 
white-of-egg.  Passive  anaphylaxis,  thus  quickly  induced, 
disappears  more  quickly  than  the  active  form,  namely 
in  about  a  fortnight.  The  mixture  of  the  anaphylactic 
serum  of  the  rabbit  sensitized  to  white-of-egg,  with  white- 
of-egg,  produced  a  precipitate.  This  was  collected  and 
diluted  in  normal  saline,  and  injected  into  the  jugular 
vein  of  two  fresh  (non-sensitized)  guinea-pigs,  and  produced 
no  reaction.* 

Whether  active  or  passive  anaphylaxis  was  conferred, 
the  injection  of  a  minute  dose  intravenously  or  intra- 
cerebrally  produced  the  classical  symptoms  and  shock 
resulting  in  death,  described  in  the  anaphylaxis  due  to 
serum  and  milk.  Injected  intraperitoneally,  it  is  rare  to 
get  grave  symptoms ;  subcutaneously,  no  anaphylactic 
symptoms  have  been  noted.  This  is  markedly  different 
from  serum  anaphylaxis,  where  the  subcutaneous  route 
is  as  fatal  as  the  others.  From  this  Besredka  and 
Bronfenbrenner  deduce  that  the  most  important  thing  in 
the  production  of  the  anaphylactic  shock  is  the  rapidity 
with  which  the  antibody  in  the  blood  comes  into  contact 


IMMUNITY    AND    ANAPHYLAXIS  217 

with  the  antibody-producer  or  antigen.  White-of-egg 
being  a  viscous  liquid  is  slowly  absorbed  from  the  peri- 
toneum or  subcutaneous  spaces,  and  so  the  combination 
of  the  antibody  and  antigen  has  not  the  suddenness  or 
instantaneousness  which  follows  intravenous  injection. 
(This  deduction  suggests  that  the  antigen  may  act  in  a 
catalytic  manner,  causing  an  amount  of  chemical  action 
out  of  proportion  to  its  own  mass  injected.  In  such  a 
case,  the  symptoms  may  be  partly  due  to  the  accom- 
paniments of  rapid  chemical  action,  such  as  the  evolution 
of  beat,  etc.) 

Antianaphylaxis  can  be  produced  to  white-of-egg,  as 
for  serum,  by  the  method  of  injecting  small  doses  during 
the  "  incubation  "  time  of  anaphylaxis  ;  or  by  the  injection 
of  a  minute  dose,  followed  by  a  larger  dose  in  10  minutes, 
and  so  on  for  four  doses.  A  guinea-pig,  previously  passively 
sensibilized,  had  o-qVo  cx-  injected  intravenously ;  in 
10  min.  another  injection  of  ^^  c.c.  ;  in  another  10 
minutes,  a  third  injection,  this  time  of  -fa  c.c.  ;  and  finally 
one  of  J  c.c.  Thereafter  the  injection  of  2  c.c.  of  white-of- 
egg,  non-diluted,  caused  uneasiness  but  nothing  further. 
Antianaphylaxis  can  also  be  established  by  oral  and  by 
rectal  feeding,  in  two  to  "four  days.  The  state  of  anti- 
anaphylaxis is  not  so  lasting  as  in  the  case  of  serum  ;  it 
lasts  about  three  weeks,  and  two  weeks  where  obtained 
by  the  oral  or  rectal  method. 

Besredka  and  Bronfenbrenner  also  found  that  the 
reactions  were  strictly  specific,  and  that  the  anaphylaxis 
produced  by  white-of-egg  was  in  the  main  specific.  Feeble 
reactions  were  given  by  the  white-of-egg  of  pigeon  and 
turtle-dove.  The  anti-anaphylactic  is  strictly  specific. 
Similar  experiments  were  made  with  heated  white-of-egg, 
with  like  results.  The  one  protects  little  against  the  other, 
so  that  they  conclude  that  their  chemical  constitution  is 
different. 

III.  Serum-Globulin. — 

Turro  and  Gonzales  have  investigated  the  subject  of 
anaphylaxis  to  determine  the  nature  of  the  substance 
which  causes  anaphylaxis  with  blood  serum.  The 
globulins  were  precipitated  from  horse's  serum,  and  the 


218         PUBLIC    HEALTH    BACTERIOLOGY 

residual  serum  was  kept.  Guinea-pigs  were  injected  with 
i  c.c.  of  a  033  per  cent  solution  of  the  globulins  ;  and 
12  days  after,  it  was  found  that  the  minimal  test  dose  was 
1  c.c.  of  a  o-66  per  cent  solution  (=  2  c.c.  of  the  former 
solution).  Anaphylactic  shock  developed  rapidly,  and  there 
was  a  rapidly  ascending  paralysis,  beginning  in  the  hind 
limbs  and  causing  death  through  asphyxia  when  the 
respiratory  centres  became  affected,  in  2  to  4  minutes. 
There  were  no  convulsions,  but  in  the  male  animals  there 
was  an  abundant  emission  of  serum. 

Another  set  of  fresh  guinea-pigs  were  given  1  c.c.  of  a 
1  per  cent  dilution  of  the  globulin-free  serum  ;  and  in 
twelve  days  thereafter,  a  test  dose  of  the  globulin  solution 
produced  no  symptoms  of  anaphylaxis.  (No  mention  is 
made  of  a  test  with  the  globulin-free  serum  itself.)  An 
animal  injected  with  normal  serum,  and  later  with  the 
globulin  solution  test  dose,  showed  intense  muscular 
tremors,  (the  animal  jumping  about),  but  all  ended  in 
recovery. 

When  the  blood  of  a  sensitized  guinea-pig  is  mixed 
with  globulin  solution,  and  1  c.c.  injected  into  the  jugular 
vein  of  a  normal  guinea-pig,  the  animal  dies  in  2  to  3 
minutes  with  symptoms  identical  with  those  described 
in  animals  sensitized  with  pure  globulins.  They  proceeded 
to  study  the  anaphylactic  poison,  which  they  found  is 
readily  destroyed  by  oxidation  and  light,  is  dialysable, 
is  thermostabile,  and  is  soluble  in  alcohol  and  ether.  They 
conclude  that  it  is  alkaloidal  in  nature,  and  should  be 
considered  a  leucomaine,  that  is,  a  toxic  substance  produced 
in  the  living  body  by  proteid  metabolism,  and  not  by 
bacterial  action. 

IV.  Classification. — 

The  foregoing  resume  of  the  subject  of  anaphylaxis 
shows  that  it  can  be  classified  in  the  same  manner  as 
immunity  into  : — 

1.  Natural  Anaphylaxis. 

2.  Acquired  Anaphylaxis. 

And  each  of  these  into  sub-groups,  thus : — 


IMMUNITY    AND    ANAPHYLAXIS  219 

Natural  anaphylaxis  depends  on — 

a.  Species  of  animal,  e.g.,  cholera  in  man  ;    anthrax 

in  cattle  ;    glanders  in  horses. 

b.  Age,    e.g.,    diphtheria   in   children  ;     erysipelas   in 

elderly  individuals. 

c.  Individual,  e.g.,  to  white  of  egg,  blood  serum  even 

by    ingestion.      ("  One    man's    food    is   another 
man's  poison.") 

Acquired  anaphylaxis  depends  on — 

a.  An    attack    of   the    disease,    e.g.,    erysipelas    and 

diphtheria. 

b.  The  injection  of  dead  cells,  e.g.,  tuberculin. 

c.  The  injection   of  nitrogenous  matter,   e.g.,   blood 

serum  and  white  of  egg. 

The  subject  of  anaphylaxis  has  been  dealt  with  here 
because  of  its  intrinsic  importance,  and  because  it  would 
seem  to  demand  a  restatement,  in  the  near  future,  of  the 
philosophy  not  only  of  infection  but  of  medicine  generally. 
The  old  idea  of  "  diathesis  "  acquires  anew  its  importance 
(having  received  direct  experimental  proof),  and  many 
apparently  worn-out  theories  and  old-fashioned  explana- 
tions of  disease  may  have  new  life  put  into  them. 


CHAPTER     XII. 

MICROCOCCI. 

These  organisms  consist  of  cells  more  or  less  globular  in 
form  and  varying  in  size  from  0-5  micron  to  2  micra  in 
diameter,  but  most  measure  about  1  micron  (TTrVo  mm.,  or 
■2 5 thro  °*  an  inch)-  They  are  usually  classified  according 
to  the  mode  of  division  or  the  resultant  shape.  Thus 
we  have  streptococci,  staphylococci,  diplococci,  tetracocci 
(tetragenus),  and  sarcinae.  None  show  endogenous  spore 
formation,  but  of  some  it  is  alleged  that  they  form  arthro- 
spores.  Most  are  non-motile,  but  a  few  motile  species 
possessing  flagella  have  been  described  (none  pathogenic). 

STAPHYLOCOCCI. 

These  were  first  demonstrated  in  pus  by  Pasteur  in 
1880  and  Ogston  in  1881,  and  in  pure  culture  by 
Becker  in  1883.  Rosenbach  in  1884  established  specificity 
as  cause  of  some  forms  of  wound-suppuration  and  of 
osteomyelitis.  They  are  so  named  from  their  growth  in 
grape-like  clusters.  Several  hundred  species  have  been 
described,  but  the .  chief  varieties  are  :  Staphylococcus 
pyogenes  (aureus,  albus,  and  citreus)  ;  Staphylococcus 
epidermidis  albus  ;  Staphylococcus  cereus  (albus  and 
flavus). 

The  common  characteristics  are  :  Grape-like  clusters  of 
cocci,  0-9  micron  in  diameter ;  non-motile  ;  non-sporing  ; 
grow  readily  on  most  media ;  stain  readily ;  Gram-positive  ; 
gelatin-liquefying  ;  produce  acid  and  clot  in  milk  ;  form 
indol ;  reduce  nitrates  to  nitrites  ;  show  colour  reduction 
with  litmus,  methylene-blue,  and  rosanilin  (fuchsin)  ; 
aerobes,  but  facultative  anaerobes ;  optimum  temperature 
for  growth,  280  to  300  C.  Range  from  8°  to  420  C. 
Thermal  death-point  30  min.  at  8o°  C. ;  freezing  useless. 

Cultures. — Put  up  (1)   broth,   (2)   agar  slope,   (3)   agar 


MICROCOCCI  221 

plate,  (4)  gelatin  stab  plate  and  slope,  (5)  milk,  (6)  potato, 
(7)  peptone  water,  (8)  nitrate,  and  (9)  sugar  media. 

In  broth,  uniform  turbidity  with  thin  surface,  pellicle 
ultimately  settling  as  a  heavy  mucoid  deposit,  with  a  sour 
odour  like  weak  butyric  acid. 

On  agar  slope,  abundant  growth  in  24  hours  at  370  C. ; 
with  smooth  shining  surface  and  resembling  a  streak  of 
oil  paint.     Single  colonies  are  circular. 

On  agar  plate,  numerous  small,  shining,  pin-head  shaped 
colonies ;  round,  finely  granular,  with  smooth  edges, 
remaining  discrete,  and  varying  greatly  in  size. 

On  gelatin  plate,  growth  occurs  readily  at  200  C,  and 
shows  much  the  same  as  in  agar.  The  colonies  are  not 
flat,  but  rise  from  the  surface  as  the  segment  of  a  sphere. 
Liquefaction  of  the  gelatin ;  and  gradually  (after  48  hours 
or  more),  shallow  saucer-shaped  depressions  are  formed, 
which  grow  larger  and  finally  become  confluent.  Lique- 
faction of  gelatin  by  staphylococci  is  due  to  a  ferment-like 
body  elaborated  by  them  and  spoken  of  as  "  gelatinase  " 
and  which  can  be  obtained  apart  from  the  cocci  by 
filtration  of  cultures.  It  is  an  extremely  thermolabile 
substance. 

In  gelatin  stab,  a  streak  of  growth  is  visible  in  24  hours, 
and  liquefaction  begins  at  the  top  in  2  to  3  days,  forming 
a  funnel  with  flocculent  deposit  of  the  bacteria.  Ultimately 
fluidification  extends  to  the  wall  of  the  tube. 

In  milk,  coagulation  takes  place  in  3  to  4  days  with 
formation  of  lactic  and  butyric  acids. 

On  potato,  growth  is  abundant,  rather  dry,  and 
usually  deeply  pigmented. 

In  peptone  water,  indol  is  formed. 

Pigment  formation  is  best  seen  in  serum  or  starchy 
media  and  aerobically  only.  It  is  insoluble  in  water  but 
soluble  in  alcohol,  chloroform,  ether,  and  benzol.  It  is 
a  C,  H,  and  O  compound,  a  "  lipochrome  ",  or  fatty  pig- 
ment.    Strong  H2S04  changes  it  to  green  or  green-blue. 

Toxic  Products. — Endotoxins,   hemolysins,    leucocidins. 

Pathogenicity. — For  man  :  abscesses,  boils,  carbuncles, 
endocarditis,  osteomyelitis ;  for  animals :  rabbits  most, 
mice  medium,  guinea-pigs  least. 

Immunization. — Take  three-weeks-old  culture,  heat  at 

1 


222         PUBLIC    HEALTH    BACTERIOLOGY 

6o°  C.  for  one  hour ;  inject  ioo  to  250  million  of  the  dead 
staphylococci,  repeating  in  3  to  4  days  later ;  opsonins 
increased. 

Habitat. — Skin  and  mucous  surfaces. 

Differentiation:  (Gordon).  —  Staphylococcus  pyogenes 
aureus  clots  milk,  liquefies  gelatin,  at  times  produces 
green  fluorescence  in  neutral  red  broth,  reduces  nitrates 
to  nitrites,  produces  acid  in  Lemco  media  containing 
maltose,  lactose,  glycerin,  and  mannite.  Staphylococcus 
epidermidis  albus  gives  the  same  reactions,  except  that  it 
does  not  produce  acid  with  mannite. 

Houston's  Lemco  Medium  :  consists  of  distilled  water 
containing  1  per  cent  of  Lemco,  1  per  cent  of  peptone, 
o-i  per  cent  of  sodium  bicarbonate,  and  litmus  solution 
to  colour.  Add  1  per  cent  of  test  substance  (carbo- 
hydrate, etc.). 

STREPTOCOCCI. 

Ogston  in  1881  first  differentiated  between  the 
irregularly  grouped  staphylococci  and  the  chain  cocci 
or  streptococci.  Pure  cultures  were  first  obtained  by 
Fehleisen  in  1883  and  Rosenbach  in  1884.  Named  from 
tendency  to  form  chains,  and  so  the  group  includes 
micro-organisms  which  differ  considerably  from  each 
other  in  cultural  and  pathogenic  characters.  The  strep- 
tococci pathogenic  to  man  mostly  form  chains  of  eight 
or  more  individual  cocci,  while  the  saprophytic  varieties 
are  apt  to  be  united  in  shorter  groups.  On  this  basis 
streptococci  have  been  divided  into  S.  longi  and  S.  breves, 
but  the  distinction  is  not  a  reliable  one.  Similarly, 
the  streptococcus  of  ordinary  pus  formation  was  thought 
to  be  different  from  that  of  erysipelas.  This  is  now  proved 
not  to  be  the  case,  and  the  two  are  regarded  as  one  and  the 
same.  These  statements  enable  one  to  attach  the  proper 
significance  to  the  various  names  used  ;  to  wit :  S.  pyogenes, 
S.  erysipelatis,  S.  longus,  S.  brevis.  S.  conglomeratus  is  a 
variety,  and  is  so  called  from  its  forming  in  broth  culture 
minute  granules  composed  of  very  long  chains  (identical 
with  S.  anginosus  or  scarlatinae) .  Streptococci  grow  well 
on  all  the  richer  media,  and  better  in  broth  made  from  meat 
or   veal    than    from    meat    extract.     Animal    serum    and 


MICROCOCCI  223 

glucose  render  media  more  favourable  for  streptococci 
cultivation,  and  alkalinity  0-5  per  cent. 

Streptococci  are  easily  stained  with  the  usual  aniline 
dyes  and  are  Gram-positive.  They  are  non-sporing,  non- 
motile,  and  non-flagellar.  They  (at  least  the  pyogenic 
species)  do  not  liquefy  gelatin.  Optimum  temperature 
37-5°  C.  Growth  takes  place  at  150  to  200  C.  (distinction 
from  pneumococci).  Aerobiosis  is  the  suitable  environ- 
ment for  most  races,  but  strict  anaerobiosis  does  not  prevent 
development  in  suitable  media.     Size  0-5  to  1  micron. 

Cultures. — In  broth  (bouillon)  minute  granules,  which 
fall  to  the  bottom  and  form  a  sparse  powdery  or  sandy 
deposit.  Diffuse  clouding  is  rare.  The  long  chain  forms 
produce  the  coarser  granules. 

In  glucose  broth,  the  rapid  formation  of  lactic  acid 
arrests  development  (1  per  cent  of  sterile  CaC03  obviates). 

In  gelatin  stab,  in  48  hours  a  thin  line  forms  which 
later  is  seen  to  be  made  up  of  minute  rounded  colonies 
of  whitish  colour  reaching  the  size  of  a  pin's  head  in  5 
to  6  days.     No  growth  on  surface  nor  liquefaction. 

On  agar  plates,  the  colonies  are  small,  greyish,  and 
delicately  opalescent,  are  round,  and  tend  to  remain 
separate  in    stroke  cultures. 

In  milk,  ready  growth  with  acid  formation  but  no  clot 
{in  pyogenic  varieties). 

On  potato,  no  growth. 

On  blood  agar  plates,  most  cause  haemolysis  and  decolor- 
ization  (difference  from  pneumococci).  Ferment  lactose, 
saccharose,  and  salicin ;   not  inulin  in  Hiss's  medium. 

Thermal  Death-point :   10  minutes  at  540  C. 

Virulence  varies ;  greatest  for  white  mice   and  rabbits. 

Habitat :  skin  and  mucous  membranes. 

Diseases :  cellulitis,  erysipelas,  osteomyelitis,  bronchitis, 
pneumonia,  empyema,  pericarditis,  otitis,  pharyngitis, 
tonsillitis,  septic  endocarditis,  septicaemia,  puerperal  fever. 

Toxins:  streptolysin  (hemolytic). 

Immunization  :    variable  results. 

Differentiation  :  (Andrews  and  Horder) . — Streptococcus 
pyogenes  does  not  clot  milk,  nor  give  green  fluorescence 
with  neutral  red  broth,  nor  produce  acid  in  Lemco  media 
with  rafhnose,  inulin,  coniferin,  and  mannite.     It  produces 


224         PUBLIC    HEALTH    BACTERIOLOGY 

acid  with  lactose,  saccharose,  and  at  times  with  salicin  ; 
it  grows  on  gelatin  at  20°  C.  ;  it  forms  long  chains,  and 
is  pathogenic  to  mice. 

Streptococcus  salivarins  clots  milk,  produces  acid  with 
saccharose  and  lactose,  and  at  times  with  raffinose ;  at 
times  grows  on  gelatin  at  20°  C,  and  at  times  produces 
fluorescence.  It  is  negative  to  the  other  tests,  and  grows 
in  short  chains. 

Streptococcus  anginosus  gives  the  same  reactions  as 
S.  salivarius,  except  that  it  never  produces  acid  with 
rafhnose  ;  it  forms  long  chains,  and  it  is  pathogenic  to  mice. 

Streptococcus  fczcalis  (human)  is  positive  to  all  the 
tests  except  production  of  acid  with  raffinose  and  inulin. 
It  is  non-pathogenic  to  mice,  and  it  forms  short  chains. 

Streptococcus  equinus  (in  horse  dung)  produces  acid 
with  saccharose,  salicin,  and  coniferin,  and  grows  on 
gelatin.  It  is  otherwise  negative  ;  forms  short  chains,  and 
is  non-pathogenic  to  mice. 

The  pneumococcus  forms  short  chains,  is  pathogenic  to 
mice,  clots  milk,  forms  acid  in  lactose,  saccharose,  and 
raffinose,  and  at  times  in  inulin.  It  does  not  grow  on 
gelatin  at  20°  C. 

Examination  of  Pus. — By  using  solid  media  and 
method  of  3  dilutions. 

(1)  Gelatin  plates  :  Three  tubes  are  melted  at  300  to 
350  C.  in  a  water-bath.  Inoculate  one  with  a  loopful 
of  pus,  replug,  and  mix  by  rotating  tube.  This  is  tube 
of  1st  dilution.  Inoculate  second  tube  with  3  loopfuls 
from  1st  tube :  tube  of  2nd  dilution.  Inoculate  third 
tube  with  3  loopfuls  from  2nd  tube  :  tube  of  3rd  dilution. 
Now  pour  out  all  three  tubes  into  dry  sterile  Petri  dishes. 
Allow  to  set,  and  incubate  at  200  C. 

(2)  Agar  plates  :  Same  procedure,  only  melt  at  1000  C. 
and  cool  to  400  C.  before  inoculating,  and  work  quickly. 
Warm  the  plates.     Incubate  at  370  C. 

PNEUMOCOCCUS. 

The  pneumococcus  has  been  at  various  times  called 
Streptococcus  pneumoniae,  Diplococcus  pneumoniae, 
Micrococcus   lanceolatus,    and   Fraenkel's   pneumococcus. 


MICROCOCCI  225 

The  sputum  of  cases  of  acute  lobar  pneumonia,  injected 
into  mice  or  rabbits,  produced  more  constantly  a  septi- 
caemia with  fatal  results  than  did  the  sputum  of  healthy 
individuals.  In  this  "  sputum  septicaemia,"  lance-shaped 
cocci  in  pairs  were  most  frequently  found.  Weichselbaum 
in  1886  examined  129  cases  of  all  forms  of  pneumonia, 
and  described  four  organisms  which  he  found,  the  most 
frequently  present  being  the  one  now  known  as  the  pneumo- 
coccus.  It  was  present  in  all  forms.  The  Streptococcus 
pneumoniae  (now  believed  to  have  been  a  more  vigorous 
pneumococcus)  was  next  in  frequency,  then  the  pneumo- 
bacillus,  and  lastly  the  Staphylococcus  pyogenes  aureus. 

Description. — A  small  coccus,  generally  occurring  in  pairs, 
and  surrounded  by  a  definite  capsule.  The  cocci  are  lance- 
shaped,  like  the  flame  of  a  candle;  and  in  the  pair  have 
the  pointed  ends  opposed.  A  close  resemblance  to  a 
bacillus  is  thus  formed.  The  capsule  is  characteristic  in 
preparations  from  the  sputum  and  tissues,  but  is  only  got 
in  serum  cultures,  and  is  absent  at  times  even  in  the 
sputum  and  in  scrapings  from  the  lung.  The  coccus  is 
non-motile,  non-flagellar,  non-sporing.  It  is  readily 
stained  by  the  usual  aqueous  aniline  dyes,  and  it  is  Gram- 
positive.  The  capsule  is  well  shown  in  specimens  stained 
by  Gram  and  counter-stained :  it  takes  the  latter. 

Cultures. — Growth  on  the  ordinary  media  is  variable. 
From  sputum  it  is  best  isolated  by  animal  injection  and 
cultures  from  the  heart's  blood.  The  best  temperature  is 
37-5°  C.  Growth  does  not  take  place  as  a  rule  below  250  C. 
The  colonies  are  like  drops  of  dew.  Blood  serum  and  blood 
agar  are  the  best  media.  In  gelatin  stab  (when  growth 
takes  place),  a  row  of  minute  dots  appears,  but  no  lique- 
faction. In  broth,  a  slight  turbidity  results,  which  settles 
to  the  bottom  of  the  tube  as  a  dust-like  deposit.  Cultures 
rapidly  die  out,  and  virulence  is  quickly  lost.  In  milk, 
growth  is  rapid,  with  acid  and  clot,  and  capsules  are 
usually  formed.  Prolonged  life  is  said  to  have  been  got  in 
cultures  on  ascitic  agar  and  blood-smeared  agar.  The 
pneumococcus  ferments  saccharose,  lactose,  ramnose,  and 
inulin. 

Habitat. — The  mucous  membranes  of  the  mouth,  nose, 
throat,  and  conjunctiva. 

15 


226         PUBLIC    HEALTH    BACTERIOLOGY 

Products. — No  soluble  toxin  yet  isolated  ;  endotoxin 
by  freezing  and  grinding.' 

Pathogenicity. — For  mice  and  rabbits,  very  great ;  for 
guinea-pigs,  less ;  for  man,  medium.  Susceptibility  is 
characterized  by  general  septicemic  infection  ;  resistance 
by  occurrence  of  localized  processes,  e.g.,  in  man :  acute 
lobar  pneumonia,  pleurisy,  empyema,  pericarditis,  menin- 
gitis, otitis  media,  etc. 

Isolation. — Inoculate  a  mouse  at  the  root  of  the  tail  with 
a  little  of  the  suspected  material.  The  animal  dies  in  24 
hours,  and  its  blood  is  swarming  with  typical,  encapsuled 
lance-shaped  diplococci,  if  pneumococci  present. 

Immunity. — Agglutinins  are  formed,  and  agglutination  is 
observed  in  1-40  to  1-50  dilution  with  serum  from  pneu- 
monic patient,  and  most  marked  at  time  of  crisis.  Passive 
immunization  with  sera  has  been  unreliable  so  far.  A 
leucocyte  extract  with  water  has  given  encouraging  results. 

Resistance. — Has  been  found  alive  in  dried  sputum 
after  55  days.  Ten  minutes  at  520  C.  is  fatal.  Very 
sensitive  to  weak  solutions  of  disinfectants,  but  not  in 
sputum. 

STREPTOCOCCUS     MUCOSUS. 

This  organism  has  been  isolated  from  cases  of  menin- 
gitis, peritonitis,  phlebitis,  and  parametritis,  and  from 
certain  cases  of  pneumonia.  It  shows  a  marked  tendency 
to  form  chains,  but  often  appears  in  diplococcus 
forms.  It  is  also  capsulated,  but  never  lance-shaped.  It 
reacts  to  sugars  as  does  the  pneumococcus.  It  is  patho- 
genic to  white  mice,  but  not  so  markedly  to  the  rabbit  as 
the  pneumococcus.  Growths  are  similar.  It  excites  the 
formation  of  weak  agglutinins,  which  can  also  in  some 
cases  agglutinate  pneumococci.  Also  anti-pneumococcic 
serum  frequently  agglutinates  it.  These  facts  suggest  a 
group  relationship. 

MENINGOCOCCUS. 

First  described  by  two  Italian  observers  in  1884, 
but  first  cultivated  by  Weichselbaum  from  cases  of 
cerebrospinal  meningitis  in  1887.  It  is  a  small  coccus, 
very  like  the  gonococcus,  and  like  it  occurring  in  pairs, 


MICROCOCCI  227 

the  adjacent  sides  being  flattened  like  a  coffee  bean 
or  two  D's  opposed  to  each  other  by  the  flat  sides. 
In  most  cases  it  is  present  inside  the  protoplasm  of 
the  leucocytes  in  the  exudation,  the  leucocytes  being 
of  the  polymorphonuclear  variety.  Hence  it  has  been 
called  the  Micrococcus  intracellularis  meningitidis.  The 
variety  of  meningitis  in  which  it  is  chiefly  found  is 
epidemic  cerebrospinal  meningitis,  which  is  now  a 
notifiable  disease  in  a  large  number  of  districts.  It  is 
non-motile,  non-sporing,  non-flagellar,  non-capsulated, 
and  Gram-negative.  It  is  found  in  the  exudates,  and 
specially  in  the  spinal  fluid  obtained  by  lumbar  puncture, 
and  in  such  fluid  it  is  well  demonstrated  in  the  cells  by 
using  Jenner's  stain.  It  is  easily  stained  by  the  usual 
dyes,  and  with  methylene-blue  stains  irregularly.  It  is 
not  readily  cultivated  on  the  ordinary  media,  and  grows 
best  on  blood  serum  and  ascitic  agar.  On  blood  serum, 
white  "shining  viscid  colonies  appear  in  24  hours.  Cultures 
readily  die  out.  It  ferments  maltose  and  dextrose  with 
acid  production  (distinction  from  M.  catarrhalis) .  Involu- 
tion forms  occur.  In  the  patient,  agglutinins  and  opsonins 
are  formed.  Distinguish  from  M.  catarrhalis,  which  is  found 
in  the  nose,  and  grows  at  room  temperature,  whereas  M. 
meningitidis  is  not  easily  grown  below  250  C.  The  sugar  test 
is  also  helpful.  M.  pharyngis  also  grows  at  room  temperature 
and  ferments  maltose,  dextrose,  saccharose,  and  laevulose. 
M.  mucosus  gives  slimy  growths.  Other  Gram-negative 
cocci  are  chromogenic. 

GONOCOCCUS. 

First  found  in  urethral  pus  and  pus  from  ophthalmia 
neonatorum  by  Neisser  in  1879.  Cultivated  by  Bumm 
in  1885  on  human  blood  serum.  Diploforms  very 
similar  to  the  meningococcus,  and  like  it  may  be  found 
in  pus,  intra-  but  also  extra-cellularly.  The  gonococci  are 
non-motile,  etc.,  and  Gram-negative.  They  are  mostly 
extra-cellular  in  chronic  discharges,  and  may  be  rather 
scarce.  They  do  not  grow  on  gelatin  or  agar,  but  on  serum 
or  ascitic  agar,  and  best  at  blood  heat.  Growth  ceases 
below  300  C.  Colonies  appear  within  48  hours,  but  may 
not  until  four  days.     They  are  readily  killed  at  420  C.     They 


228 


PUBLIC    HEALTH    BACTERIOLOGY 


ferment  dextrose  but  not  maltose.  A  vaccine  treatment 
lor  gonococcus  has  been  used  with  success  in  chronic  cases. 
An  emulsion  of  gonococci  in  sterile  salt  solution  (0-85  per 
cent)  is  heated  to  650  C.  for  1  hour.  Inject  300  million, 
and  give  dose  every  7  to  10  days,  increasing  to  1000  to 
1200  million. 

MICROCOCCUS  TETRAGENUS. 

Found  in  1881  by  Gaffky,  in  pulmonary  cavities. 
Usually  non-pathogenic  in  man.  Grows  well  on  ordinary 
media,  clouds  broth  evenly,  does  not  liquefy  gelatin,  clots 
milk  with  acid  formation,  is  Gram-positive,  and  is  cap- 
sulated  in  the  body.  It  is  pathogenic  to  white  mice, 
causing  septicaemia  ;  slightly  so  to  guinea-pigs  and  rabbits, 
and  non-pathogenic  to  house  mice  and  rats.  It  has  been 
described  as  the  cause  of  abscesses,  on  one  occasion  of 
meningitis,  and  on  another  of  septicaemia. 

MICROCOCCUS    CATARRHALIS. 

Found  in  patients  suffering  from  catarrh  of  the  upper 
respiratory  tract.  Its  chief  claim  to  attention  is  its 
similarity  in  staining  and  morphology  to  the  meningo- 
coccus and  gonococcus.  From  the  latter  it  is  distinguished 
by  its  rapid  growth  on  the  ordinary  culture  media.  From 
the  meningococcus,  with  which  it  may  be  found  in  the 
nasal  passages,  it  is  similarly  distinguished,  but  here  the 
difference  is  one  of  degree  only.  The  sugar  tests  are  very 
helpful. 


Dextrose 

Maltose 

Laevulose 

Sacchar- 
ose 

Lactose 

Galactose 

Meningococcus  . . 
Gonococcus 
M.  catarrhalis    . . 
M.  pharyngis  sice. 

+ 
+ 
O 

+ 

+ 
O 
O 

+ 

O 
O 
O 

+ 

O 
O 
O 

+  . 

O 
O 
O 
O 

O 
O 
O 
O 

M 

ICROC 

DCCUS 

MELI 

TENSI! 

3. 

This  organism  was  first  described  by  Bruce  in  1887, 
as  present  in  the  spleen  of  patients  dead  of  Malta  (or 
undulant,    rock,    Mediterranean,    or    Neapolitan)    fever. 


MICROCOCCI  229 

This  is  an  endemic  pyrexial  disease,  occasionally  pre- 
vailing as  an  epidemic,  having  a  long  and  indefinite 
duration  and  an  irregular  course,  with,  almost  invariably, 
pyrexial  relapses  of  an  undulatory  type.  The  illness 
may  last  from  20  to  300  or  more  days,  averaging  60  to 
70  days,  and  having  a  mortality  of  about  2  per  cent. 
The  chief  mode  of  spread  formerly  was  the  ingestion  of 
goat's  milk,  in  which  the  micrococcus  was  being  excreted. 
The  urine  of  patients  is  also  infectious.  The  Mediterranean 
Fever  Commission  conclude  that  Malta  fever  is  a  septi- 
caemia, in  which  the  specific  organism  can  be  recovered 
from  the  peripheral  blood,  the  urine,  and  the  faeces. 
Infection  is  not  conveyed  by  the  sputum,  sweat,  breath, 
or  skin-scrapings  of  patients.  It  does  not  take  place  if 
contact  is  limited  to  skin  surfaces  only,  and  if  urinary  and 
faecal  contamination  are  excluded.  It  is  probably  occasion- 
ally conveyed  by  sexual  intercourse.  About  10  per  cent  of 
the  Maltese  goats  excrete  the  micrococcus,  and  50  per  cent 
give  a  positive  agglutination  reaction.  Goat's  milk  is  prob- 
ably the  prime  source  of  the  disease  in  most  cases  if  not  in  all. 

Characteristics. — Micrococcus  melitensis  is  a  very  small 
coccus,  0-3  micron  in  diameter,  occurs  singly  or  in  pairs, 
and  at  times  in  short  chains.  It  is  non-motile  and  Gram- 
negative  ;  does  not  ferment  glucose  (unlike  ordinary  strepto- 
coccus), and  renders  milk  slowly  alkaline.  It  is  easily 
stained.     Some  observers  describe  it  as  a  minute  bacillus. 

Cultures. — On  agar,  it  forms  minute  transparent  colonies 
likened  to  dew-drops,  but  only  after  2  to  3  days'  culture  at 
37-5°  C.  On  gelatin,  liquefaction  is  not  produced.  In 
broth,  no  indol  is  formed,  nor  odour ;  but  a  slight  turbidity. 
On  potato,  a  moist  transparent  growth  is  formed. 

Agglutination  of  the  organism  by  the  patient's  serum 
is  shown  after  the  fifth  day.  In  some  cases  it  persists  for 
years. 

Resistance. — Is  a  vigorous  organism  and  resists  desicca- 
tion for  weeks. 

Malta  Fever. — Other  symptoms  of  the  disease  are : 
shifting  rheumatic-like  pains,  profuse  sweatings,  con- 
stipation, local  neuritis,  emaciation,  and  almost  always 
enlarged  spleen.  The  micrococcus  may  be  isolated  by 
splenic  puncture  or  from  the  blood. 


CHAPTER    XIII. 

NON-SPORING    BACILLI. 

THE    COLON-TYPHOID-DYSENTERY   GROUP. 

This  is 'a  large' group,  which  includes  the  colon  bacillus 
and  its  allies  ;  the  typhoid  bacillus  ;  paratyphoid  bacilli ; 
dysentery  bacilli  and  allies  ;  and  Bacillus  faecalis  alcaligenes. 

Closely  related  to  the  group  (but  not  properly  within  it) 
are  the  B.  lactis  aerogenes,  B.  mucosus  capsulatus  (Fried- 
laender's  bacillus),  and  B.  proteus. 

\  All  the  members  of  the  group  are  bacilli  and  are  very 
similar  morphologically,  but  exhibit  minor  differences 
insufficient  to  permit  of  accurate  diagnosis  from  mor- 
phology alone.  All  are  non-sporing,  non-liquefying  of 
gelatin,  Gram-negative,  and  grow  well  at  room  and  body 
temperatures  on  artificial  media. 

They  are  distinguished  from  one  another  by  a  careful 
cultural  and  biological  study,  to  wit :  reactions  in  special 
media  (e.g.  the  fl-ag-in-ac  group  of  reactions : — fluor- 
escence with  neutral  red,  acid  and  gas  with  lactose,  indo] 
with  peptone  water,  acid  and  clot  in  litmus  milk) ;  motility 
and  flagella ;  and  reactions  with  specific  immune  sera 
(chiefly  agglutination) . 

Bacillus  Coli  Communis  is  a  name  which  stands  for  a 
group  of  organisms,  one  member  of  which  was  first  described 
by  Buchner  in  1885.  The  one  taken  as  a  type  of  the  group 
was  obtained  from  the  stools  of  a  breast-fed  infant,  and 
was  described  by  Escherich  in  1886  as  the  Bacterium  coli 
commune.     It  is  now  usually  designated  B.  coli  (Escherich). 

It  is  widely  distributed  in  nature  and  has  been  isolated 
from  air,  water,  and  soil,  but  is  found  most  abundantly  and 
constantly  in  the  intestinal  tract  of  man  and  of  many  of 
the  higher  animals,  from  which  habitat  it  probably  rinds 
its  way  into  soil,  water,  and  air.  Its  chief  characteristics 
are  :  short  plump  rod,  2  to  4  micra  long  and  0-4  to  07  micron 
broad  (very  short  oval  and  coccus-like  forms  are  found, 


NON-SPORING    BACILLI  231 

especially  in  the  animal  tissues) ;  grows  well  on  usual  media  ; 
curdles  .  milk  with  acid  production  in  forty-eight  hours  ; 
forms  acid  and  gas  with  dextrose  and  lactose  (gas  = 
hydrogen  and  carbonic  acid  gas  in  proportion  of  2  to  1) ; 
some  ferment  saccharose ;  some  are  weak  and  aberrant ; 
not  usually  pathogenic  (agonal  or  post-mortem  invasion 
excluded)  ;  sometimes  causes  peritonitis,  cholecystitis, 
pyelitis,  and  cystitis ;  can  precipitate  cholesterin  from 
solution  (hence  may  cause  gall-stones)  ;  cannot  peptonize 
native  proteids  (casein  and  egg  albumen,  etc.). 

Cultures. — In  broth  :  uniform  turbidity.  In  gelatin  stab  : 
growth  along  whole  line  of  stab  and  film-like  abundant 
growth  on  surface,  but  no  liquefaction.  On  gelatin  plate  : 
surface  colonies  are  apt  to  show  the  typical  grape-leaf 
formation.  (In  gelatin  stab,  a  few  gas  bubbles  may  form, 
see  later.)  On  agar  slope  :  a  dense  glistening  white?  or 
greyish  growth.  Same  on  blood  serum.  On  agar  plate : 
surface  colonies  show  grape-leaf  structure.  On  potato : 
abundant  growth,  at  first  greyish- white,  turning  later  to 
yellowish-brown.  Cultures  are  characterized  by  a  peculiar 
foetid  odour,  not  unlike  that  of  diluted  faeces.  Grows  well 
on  media  containing  urine  and  bile.  In  peptone  water : 
forms  indol.  In  milk  :  acidity  and  clot.  In  lactose  litmus 
agar :  the  medium  becomes  red  along  stab,  and  gas  bubbles 
appear.  In  carbohydrate  media  :  acid  and  gas  are  formed 
in  presence  of  glucose,  lactose,  laevulose,  galactose, 
maltose,  raffmose,  mannite,  dulcite,  and  sorbite ;  and 
occasionally  in  saccharose  (cane  sugar)  and  in  the  gluco- 
sides,  salicin  and  arbutin.  Some  varieties  change  neutral- 
red,  first  to  a  rosy-red  and  then  to  a  green  fluorescence 
(in  glucose,  broth)  ;  and  most  reduce  nitrates  to  nitrites. 
Aerobe,  but  facultative  anaerobe.  Motile,  having  from 
4-12  peritrichal  flagella. 

B.  Typhosus  was  discovered  by  Eberth  in  1880  in  the 
spleen  and  mesenteric  glands  of  persons  dying  of  typhoid 
or  enteric  fever.  In  such  sections  the  bacilli  occur  in 
groups,  scattered  individuals  being  rare.  Gaffky  in  1884 
first  grew  it  in  pure  culture  and  studied  its  characters. 

Characteristics. — Short  plump  rod  with  rounded  ends7 
1  to  3  micra  long  and  05  to  08  broad ;  actively  motile ; 
numerous    peritrichal  flagella    (10    to    14)  ;    growth   less 


232  PUBLIC    HEALTH    BACTERIOLOGY 

luxuriant  than  B.  coli ;  Gram-negative  ;  produces  acid  but 
no  gas  in  dextrose  broth  and  agar,  no  change  with  lactose 
or  saccharose,  no  clot  in  milk,  but  in  litmus  milk  slight  acid 
at  first,  from  small  quantity  of  monosaccharid  present  ; 
later,  deep  blue  from  formation  of  alkali ;  no  indol  in 
peptone  water ;  on  •  potato  slight  moist  glistening  growth, 
becoming  dull  and  velvety. 

Cultures. — In  broth  :  uniform  turbidity.  In  gelatin  stab  : 
growth  to  bottom  of  stab,  and  on  surface  as  a  thin  leaf- 
like film  or  pellicle  with  irregular  wavy  margin.  In  gelatin 
plate,  the  colonies  are  smaller,  more  delicate,  and  more 
transparent  than  those  of  the  colon  bacillus  of  the  same 
age.  In  agar  plates,  in  twenty-four  hours,  small  greyish 
colonies  are  formed,  at  first  transparent  but  later  opaque. 
On  acid  potato :  slight  moist  glistening  growth. 

Forms  acid  but  no  gas  with  dextrose,  laevulose,  galactose, 
maltose,  mannite,  and  dextrin  ;  but  no  change  with  lactose 
and  saccharose. 

In  Hiss's  tube  medium  (agar,  gelatin,  dextrose,  Liebig's 
extract,  salt,  water),  it  gives  uniform  clouding  owing  to 
its  motility,  but  no  gas  ;  whereas  dysentery  bacillus  grows 
only  along  stab,  and  colon  bacilli  form  sky-rocket-like 
figures,  and  the  medium  is  broken  with  gas  bubbles. 

Optimum  temperature,  370  C.  ;  range  15 °  to  41  °  C. 
Resistance  like  most  non-sporing  organisms,  30  min.  at 
6o°  C.,  or  2  to  3  min.  at  ioo°  C.  In  ordinary  tap  or  distilled 
water  it  is  usually  found  dead  in  three  weeks  (Frankland). 
The  researches  of  Dr.  A.  C.  Houston  have  shown  that  in 
the  crude  river  water  derived  from  the  Thames,  raw 
typhoid  bacilli  die  out  in  one  to  two  weeks,  whereas 
cultivated  typhoid  bacilli  may  persist  for  five  weeks.  On 
this  basis,  the  storage  of  water  for  thirty  days,  preliminary 
to  filtration,  is  looked  upon  as  of  prime  importance  in  all 
waters  derived  from  sources  polluted  by  sewage. 

Does  not  multiply  in  water,  even  when  impure.  Is  by 
preference  a  parasite,  and  when  found  outside  the  body 
can  be  usually  traced  to  sewage  of  typhoid  patient  or 
convalescent  (carrier).  In  natural  bodies  of  water  it 
retains  its  vitality  for  at  least  four  to  five  days,  and  in 
sterile  water  it  may  be  found  surviving  for  three  months. 
History  of  typhoid  epidemics  from  sewage-polluted  water 


NON-SPORING    BACILLI  233 

shows  that  danger  is  chiefly  to  be  feared  when  pollution  is 
recent.  In  soil  and  faecal  matter,  however,  duration  of 
vitality  is  more  prolonged  (five  months  in  privy  refuse, 
and  fourteen  days  after  being  spread  on  ground),  but  no 
genuine  multiplication  proved.  This  suggests  one  mode 
of  pollution  of  surface  water  and  surface  wells  after  rains, 
by  the  washing  of  dormant  bacilli  into  the  same,  and  to  the 
danger  of  using  human  excrement  for  manuring  vegetable 
gardens  and  fields  near  water  sources  (Lincoln,  1905). 
Air-borne  infection  is  rare.  Sewer  air  is  not  regarded  as 
an  actual  cause  so  much  as  a  predisposing  one. 

Pathogenicity. — For  man :  enteric  fever,  typhoid  fever, 
abdominal  typhus  (German),  la  fievre  typhoide  (French). 
For  animals :  do  not  multiply,  and  same  effects  by 
injection  of  dead  bacilli,  as  that  due  to  endotoxins. 
Chimpanzees  have  been  infected  by  food,  and  have 
shown  characteristic  lesions.  Intraperitoneal  injections 
produce  a  short  acute  illness  with  pyrexia,  etc.,  but  non- 
specific. 

Specificity  has  not  been  absolutely  proved  according  to 
Koch's  postulates.  In  enteric  fever  the  bacilli  are  present 
in  the  blood,  the  bowel,  the  urine,  the  sputum  and  the 
rose  spots.     Toxins  are  intracellular. 

Immunity  usually  follows  one  attack,  and  is  due  to 
bactericidal  and  bacteriolytic  bodies. 

Active  Immunization  is  accomplished  in  animals  by  a 
first  injection  of  1  c.c.  of  a  broth  culture  heated  for  ten 
minutes  at  6o°  C,  followed  in  five  or  six  days  by  a  larger 
dose,  and  so  on,  until  finally  living  cultures  are  injected  in 
considerable  doses  without  serious  consequences. 

Wright's  vaccination  against  typhoid  has  been  used 
extensively  in  the  British  army.  He  uses  a  strain  of 
bacillus  standardized  by  passage  through  guinea-pigs,  and 
sterilizes  the  culture  by  heating  to  6o°  C.  for  five  minutes. 
The  first  injection  is  of  an  amount  of  bacilli  fatal  to  100 
grm.  of  guinea-pig,  or  alternatively,  750  to  1000  million 
of  dead  bacilli.  The  second  injection  given  eleven  days 
later  should  be  double  the  first.  The  first  dose  is  followed 
by  tenderness  and  swelling  locally  and  at  the  adjacent 
lymphatic  glands,  and  some  pyrexia,  all  of  which  usually 
subside  in  twenty-four  to  forty-eight  hours.     The  method 


234  PUBLIC    HEALTH    BACTERIOLOGY 

is  believed  to  reduce  the  case  incidence  and  the  mortality 
among  vaccinated  persons  attacked. 

Vaccine  treatment  of  typhoid  fever  on  similar  lines  has 
been  tried  by  Leishman  and  Smallman,  using' one-fifth  of 
the  dose  used  for  protective  inoculation.  If  the  tempera- 
ture fall,  the  injection  may  be  repeated  every  four  days. 
Antityphoid  serum  has  been  used,  but  results  are  equivocal. 

Agglutination. — The  blood  or  blood  serum  of  an  animal 
previously  inoculated  with  typhoid  bacilli,  when  added  to 
a  suspension  of  living  and  motile  typhoid  bacilli,  causes 
the  latter  to  become  motionless  and  aggregated  (clumped). 
This  reaction  is  one  given  in  many  diseases  after  the 
attack — a  reaction  of  immunity  ;  but  in  enteric  fever  it  is 
given  during  the  attack — a  reaction  of  infection.  The 
reaction  is  specific  in  high  dilutions,  but  not  absolutely 
so.  Group  bacilli  are  clumped,  but  in  lower  dilutions  : 
for  example,  serum  clumping  typhoid  bacilli  in  a  dilution 
1-40,000  will  clump  paratyphoid  bacilli  in  a  dilution 
1-1000,  and  coli  bacilli  in  1-500.  The  reaction  is  used 
for  :  (1)  Diagnosis  of  disease  (Widal's  reaction)  ;  and 
(2)  Diagnosis  of  bacteria  (Grtieber's  reaction). 

1.  Widal's  Reaction. 

a.  Microscopic  method. — Requirements  :  An  eighteen  to 
twenty-four  hours'  culture  in  broth  of  undoubted  B. 
typhosus.  Blood  or  serum.  Platinum  loop,  a  hollow 
ground  slide,  an  ordinary  slide  or  a  watch-glass,  cover- 
slips,  a  one-sixth-inch  lens,  and  vaseline. 

Procedure. — 1.  Test  motility  of  bacilli  and  absence  of 
clumping  by  putting  up  a  hanging  drop  of  culture.  If 
clumps,  filter.     If  movements  sluggish,  warm. 

2.  Take  a  clean  dry  slide  and  put  on  it  nine  loopfuls  of 
sterile  broth  arranged  in  a  small  circle.  Put  one  loopful 
of  serum  in  centre  of  circle  and  mix.  Dilution  1-10. 
Now  put  one  loopful  of  1-10  dilution  on  a  clean  slide  or 
hollow  slide,  mix  with  three  loopfuls  of  sterile  broth,  and 
finally  with  one  loopful  of  culture.     Dilution  is  now  1-50. 

3.  Mount  one  loopful  on  a  cover-slip,  invert  over  hollow 
slide,  and  examine.  Examine  again  in  fifteen  minutes  and 
after  one  hour.  If  reaction  is  positive,  the  bacilli  will  be 
arranged  in  groups  and  be  non-motile,  and  between  the 
clumps  will  be  clear  spaces. 


NON-SPORING    BACILLI  235 

Or  the  serum  may  be  diluted  by  means  of  a  graduated 
pipette,  such  as  a  leucocytometer  pipette,  or  by  using  a 
capillary  pipette  which  is  rilled  to  a  certain  mark  and 
emptied  into  a  watch-glass,  and  the  desired  number  of  the 
full  of  the  pipette  of  bouillon  added  and  mixed.  The 
mixture  of  a  pipette-full  of  the  diluted  serum  and  of  the 
bacillary  emulsion  is  then  examined,  the  final  dilution 
being  double  that  of  the  diluted  serum. 

b.  Macroscopic  method  or  Sedimentation. — Take  a  range 
of  test  tubes,  5  cm.  x  0-5  cm.,  and  put  into  each  1  ex. 
of  various  serum  dilutions  and  1  c.c.  of  bacillary  emulsion. 
Also  put  up  a  control  with  1  in  20  normal  serum.  Plug, 
and  keep  vertical,  and  either  incubate  for  three  hours  at 
370  C,  or  keep  at  room  temperature  for  twelve  to  twenty- 
four  hours ;  or  first  incubate  and  then  keep  for  twenty-four 
hours,  reading  the  results  at  both  stages.  The  result  can 
be  controlled  by  microscopic  examination  of  the  super- 
natant fluid. 

The  statement  of  the  result  should  comprise  all  the 
conditions  of  the  experiment,  namely :  kind  of  test  (hanging- 
drop  or  tube),  dilution  of  serum,  times  of  observation,  and 
intensity  of  reaction  (complete,  medium,  or  nil  agglutin- 
ation). 

The  test  is  also  given  by  dead  bacilli.  Young  cultures 
are  used  to  prevent  spontaneous  agglutination.  It  is  not 
always  convenient  to  have  young  cultures,  and  so  the 
following  suspension  of  dead  bacilli  (which  keeps  well  but 
agglutinates  tardily  though  easily),  may  be  used :  To  a 
twenty-hours'  broth  culture,  add  1  per  cent  formalin, 
incubate  for  two  days  at  370  C,  pour  off  the  fluid  from 
the  precipitate  and  store  in  an  ice  chest. 

Interpretation  of  Results. — A  positive  Widal  reaction 
may  be  due  to  :  (1)  An  attack  of  typhoid  fever  (especially 
if  increasing)  ;  (2)  A  previous  attack ;  (3)  An  attack  of 
paratyphoid  fever  ;  (4)  Some  other  disease  (jaundice  and 
tuberculosis) . 

A  negative  Widal  may  be  due  to  :  (1)  Too  great  or  too 
little  infection  ;  (2)  Disease  not  typhoid  ;  (3)  Test  applied 
too  early  (before  eighth  day)  ;  (4)  Inhibition  phe- 
nomena (some  sera  agglutinate  in  a  dilution  1-100  and 
not  in  1-20). 


236  PUBLIC    HEALTH     BACTERIOLOGY 

2.  Grueber's  Reaction. — Consists  in  determining  the 
race  of  a  bacterium  by  testing  it  against  the  blood  serum 
of  an  animal  immunized  to  an  organism  of  known  race. 
The  reaction  is  the  converse  of  the  Widal  one,  but  high 
dilutions  (i-iooo)  must  be  used  to  avoid  error  due  to 
group  agglutinins.  It  is  open  to  the  fallacies  that  some 
bacilli  are  inagglutinable,  though  belonging  undoubtedly 
to  an  agglutinable  race  (some  typhoid  bacilli),  and  bac- 
teria giving  a  negative  result  may  nevertheless  have 
very  similar  pathogenic  characters  to  those  giving  a 
positive  result. 

Principal  Channels  of  Infection. — Water,  milk,  ice-cream 
foods,  oysters,  mussels,  water-cress,  lettuces,  radishes,  flies, 
dust,  contact,  chronic  germ-carriers. 

Paratyphoid  Bacilli  (also  called  paracolon  bacilli) — 
are  the  probable  cause  of  a  disease  clinically  resembling 
mild  typhoid  (about  3  per  cent  of  cases  treated  as  typhoid 
are  probably  paratyphoid).  Onset  is  usually  sudden,  with 
chills;  mortality  is  low  (2  per  cent),  and  on  post-mortem 
examination  no  characteristic  ulceration  of  Peyer's  patches 
is  observed.  Two  chief  varieties  are  described,  namely, 
paratyphoid  "  A "  and  paratyphoid  "  B."  Of  these  B 
is  the  more  widely  distributed  in  nature,  and  is  more 
pathogenic  to  animals,  and  is  believed  by  some  to  be  the 
same  as  B.  enteritidis  (Aertryck)  and  B.  typhi  murium 
(mouse  typhoid) .  In  cultures  A  resembles  the  typhosus ; 
and  B  the  colon  bacillus ;  and  fermentative  reactions  like- 
wise, except  in  milk,  where  A  gives  slight  permanent  acidity, 
and  B  slight  acidity  which  after  the  third  day  gives  place 
to  alkalinity.  Cases  of  illness  due  to  A,  resemble  mild 
typhoid ;  and  to  B,  are  allied  to  food-poisoning  with 
severe  gastro-intestinal  symptoms.  Organisms  of  this 
group  form  endotoxins  which  are  heat  resisting,  and  there- 
fore the  ingestion  of  cooked  food  containing  the  bacilli 
may  produce  severe  disturbance,  thus  relating  them  to 
Gaertner's  bacillus  (B.  enteritidis).  None  form  indol. 
All  give  fluorescence  with  neutral-red.  These  bacilli  are 
motile,  flagellar,  show  agglutination  phenomena,  and 
ferment  dextrose,  dulcite  and  mannite,  but  not  lactose 
nor  saccharose.  They  are  present  at  times  with  the  B. 
typhosus,  and  may  thus  produce  a  mixed  infection. 


NON-SPORING    BACILLI  237 

B.  Enteritidis  (Gaertner).— In  1888  the  flesh  of  a 
diseased  cow  was  sold  for  food  in  a  Saxony  village.  Gastro- 
enteritis followed  the  ingestion  of  the  meat  in  the  case  of 
fifty-seven  people.  One  young  man  ate  800  grm.  (nearly 
2  lb.)  of  the  raw  meat,  and  died  in  thirty-five  hours. 
From  his  spleen  and  blood  Gaertner  isolated  an  actively 
motile  bacillus,  closely  resembling  the  typhoid  germ  ; 
and  he  obtained  the  same  organism  from  the  flesh  of  the 
cow.  Similar  bacilli  have  since  been  found  in  other 
outbreaks  of  meat-poisoning.  Gaertner's  bacillus  is  very 
pathogenic  to  laboratory  animals,  causing  an  intense 
hemorrhagic  enteritis.  The  symptoms  are  due  to  endo- 
toxins which  are  heat  resisting,  so  that  boiling  does  not 
readily  destroy  the  toxicity.  It  grows  more  rapidly  on 
gelatin  than  B.  typhosus  ;  forms  no  indol,  but  ferments 
dextrose,  with  formation  of  acid  and  gas.  Closely  related 
to  the  bacillus  of  Gaertner  are  the  hog  cholera  bacillus 
and  the  B.  psittacosis.  The  latter  was  first  isolated 
in  Paris  in  1892  in  a  highly  fatal  pneumonia-like  illness 
(49  cases,  16  deaths),  which  was  traced  to  sick  parrots 
from  South  America.  B.  icteroides  and  B.  typhi 
murium  are  also  of  this  group.  Danysz's  virus  is 
supposed  to  consist  of  B.  enteritidis  (iErtryck  and 
Gaertner) . 

B.  Dysenteriae,  or  Shiga's  bacillus.  First  isolated 
from  the  stools  of  patients  (in  Japan)  suffering  from  acute 
dysentery,  in  which  no  amoeba  could  be  found.  The 
bacillus  was  found  by  examining  the  stools  for  an  organism 
which  would  agglutinate  with  the  serum  of  the  patients. 
In  36  cases  one  and  the  same  organism  was  found  to  meet 
the  test,  and  it  was  not  found  in  the  dejections  of  healthy 
persons  or  persons  suffering  from  other  diseases,  nor  did 
it  agglutinate  with  their  blood  serum.  It  is  now  recognized 
as  the  specific  cause  of  acute  epidemic  dysentery  of 
temperate  climates.  Since  then,  several  bacilli  have  been 
isolated  in  different  parts  of  the  world,  all  related  to  Shiga's 
bacillus  but  giving  a  variety  of  reactions  to  carbohydrates 
and  to  immune  serum.  Kruse  isolated  his  organism  from 
"  pseudo-dysentery  of  the  insane,"  and  Flexner  from  dysen- 
tery in  the  Philippines.  They  all  ferment  dextrose  but 
without  gas  formation.     In  milk,  first  slight  acidity  and 


238  PUBLIC    HEALTH    BACTERIOLOGY 

then  increased  alkalinity  is  produced.  The  Shiga-Kruse 
group  produce  no  indol,  but  the  Flexner  group  do.  Short 
rod,  non-motile,  non-liquefying,  non-Gram-staining,  aerobe 
and  facultative  anaerobe. 

Bacillary  dysentery  is  an  ulcerative  colitis  with  little  ten- 
dency to  form  liver  abscess.  It  is  spread  like  enteric  fever, 
and  is  a  scourge  of  armies  as  it  formerly  was  of  asylums. 
Animals  are  easily  killed  by  injection,  but  show  no  charac- 
teristic lesions  in  the  intestine,  though  the  latter  have  been 
obtained  by  feeding  experiments.  The  Shiga-Kruse  bacilli 
form  a  soluble  toxin  which  is  very  fatal  to  rabbits,  and  is 
resistant  up  to  700  C.  This  toxin  causes  profuse  diarrhoea, 
and  later  paralysis,  when  injected  intravenously  into 
rabbits,  being  apparently  excreted  by  the  colon  and  caecum. 

Similar  bacilli  have  been  isolated  in  the  summer  diarrhoea 
of  infants. 

An  anti-toxic  serum  has  been  prepared  from  horses. 

B.  Faecalis  Alcaligenes  resembles  B.  typhosus 
morphologically  and  culturally,  but  is  non-pathogenic. 

Cultures. — Gives  a  luxuriant  growth  on  potato  ;  forms 
no  acid  with  glucose,  but  alkali  with  milk  whey  and 
mannite.  It  is  an  occasional  inhabitant  of  the  ileum  and 
colon. 

Other  organisms  which  have  been  described  in  connection 
with  the  colon-typhoid-dysentery  group  are :  B.  nea- 
politanus  (in  a  choleraic  disease)  ;  B.  acidi  lactici,  of 
Hueppe ;  B.  lactis  aerogenes  (resembles  Friedlaender's 
diplobacillus),  and  other  bacilli  present  in  milk,  which 
appear  in  the  faeces  of  milk-fed  persons. 

Voges-Proskauer  Reaction  is  not  given  by  B.  coli 
nor  by  any  of  the  above,  but  by  B.  lactis  aerogenes,  B.  cloacae, 
and  B.  oxytocus  perniciosus.  Inoculate  glucose-peptone 
solution  and  grow  for  3  days ;  add  KOH ;  stand  for 
twenty-four  hours.     Red  colour. 

Differentiation  of  B.  Typhosus  from  B.  Coll 

The  problem  is  to  find  a  medium  which  will  favour  the 
development  of  B.  typhosus  and  B.  coli  and  yet  will 
differentiate  them.  The  usual  method  is  to  use  coloured 
media  +  inhibitory  agents  or  favouring  agents. 


NON-SPORING    BACILLI  239 

1.  Drigalski  and  Conradi's  Medium. — This  is  a  meat 
broth  (1-5  lb.  per  litre)  to  which,  besides  the  peptone  and 
salt,  i  per  cent  of  mitrose  and  3  per  cent  of  agar  are  added 
and  dissolved.  Thereafter  litmus  solution  is  used  to 
dissolve  pure  lactose  (quantity  used  =  half  quantity  of 
agar) ,  and  the  whole  is  added  to  the  hot  agar  fluid.  Render 
alkaline  with  sodium  carbonate  solution,  and  add  a  solution 
of  crystal- violet.  The  medium  must  not  be  overheated  or 
the  lactose  may  be  changed.  It  is  a  solid  medium  and  is, 
shortly,  a  lactose-nutrose-agar. 

The  crystal-violet  restrains  the  saprophytes.  In 
twenty-four  hours  B.  coli  colonies  are  red  ;  2  to  6  mm.  in 
diameter,  and  non-transparent.  B.  typhosus  colonies  are 
blue  ;   2  mm.  in  diameter,  and  glassy  and  dew-like. 

The  plates  are  inoculated  by  smearing  the  surface  with 
a  glass  rod,  dipped  in,  say,  diluted  faeces. 

2.  Endo's  Medium. — This  is  a  3  per  cent  agar  neutralized 
and  then  alkalinized  with  NaOH,  and  lactose  and  fuchsin 
(basic)  solution  added,  and  then  Na2S03  solution  until 
decolorized.  Put  into  test  tubes  (15  c.c),  sterilize,  and 
keep  in  dark.  When  using,  pour  plates,  and  inoculate  by 
surface  smears,  when  in  twenty- four  hours  B.  coli  colonies 
are  red,  and  B.  typhosus  colonies  are  colourless. 

3.  Loeffler's  Medium. — This  is  a  3  per  cent  agar  to  which 
malachite-green  is  added,  and  this  retards  growth  of  B. 
coli.  B.  typhosus  colonies  are  minute  glistening  points  ; 
later,  they  colour  agar  yellow. 

4.  Hoffman  and  Ficker's  Medium. — Convert  water  sample 
into  medium  by  adding  :  caffein  2-5  per  cent  (restrains 
B.  coli)  ;  nutrose  1  per  cent ;  and  crystal-violet  o-ooi  per 
cent  (restrains  saprophytes).  Incubate  at  370  C.  for  not 
more  than  12  hours.  The  B.  typhosus  can  then  be  isolated 
on  plate  media. 

5.  MacConkeys  Media — Have  been  already  described  on 
page  153.  The  bile-salt  assists  growth  of  B.  coli  and 
B.  typhosus,  and  hinders  others.  Where  neutral-red  is 
used,  acid  formation  changes  it  to  a  rose-red. 

6.  Bile  Medium  (for  blood). 

7.  Hiss's  A  gar-Gelatin  Media,  see  p.  232. 


240 


PUBLIC    HEALTH    BACTERIOLOGY 


Table  of  Characteristics. 


B.  Coli 

B.  Typhosus 

Paratyphosus 
A        B 

Dysenteriae    jFajcalis 
A         B        I    Ale. 

Motility 

+ 

+ 

+       + 

—       — 

+ 

Gram 

negative 

negative 

negative 

negative 

neg. 

Peptone  water 

indol 

— 

—      — 

—  indol 

— 

Glucose 

A  +  G 

A 

A+GA+G 

A     A 

nil 

Lactose 

A  +  G 

— 

Saccharose 

— 

— 

—      — 

_      _     j   _ 

Milk  (litmus) 

A  +  Clot 

(  A  (alk.  in  4 

A  (alk.  in 

A  (alk.  in    |  Alk. 

!  to  10  days) 

3  days) 

3  days) 

Gelatin 

non- 

non- 

non- 

non-       1  non- 

liquefying 

liquefying 

liquefying 

liquefying  llique- 

Pathogenic : 

For  man 

slight 

distinct 

distinct 

distinct 

nil 

,,     animals 

do. 

less 

more 

more       j  nil 

Short  Table  of  Carbohydrate  Reactions. 


1 

Glucose 

Lactose 

Saccharose 

Dulcite 

Mannite 

B.  typhosus     . . 

A 

A 

B.  coli  .  . 

+ 

+ 

— 

+ 

+ 

B.  acidi  lactici   ! 

+ 

+ 

— 

— 

+ 

B.  paratyphosus 

+ 

— 

— 

+ 

+ 

A  signifies  acid  ;     +  acid   and  gas  ; 


-    nil. 


CAPSULATED     BACILLI. 

In  this  group  are  classed  bacilli  which  are  non-motile, 
and  capsular ed  in  certain  cultures,  but  otherwise  resemble 
the  Bacillus  coli. 

Bacillus  Pneumoniae.  —  Otherwise  called  pneumo- 
bacillus,  Friedlaender's  bacillus.  Was  first  described  by 
Friedlaender  in  1882  as  the  cause  of  acute  lobar  pneumonia. 
It  was  first  called  a  micrococcus,  and  was  confused  with 
Fraenkel's  pneumococcus,  but  was  later  recognized  as  a 
short  bacillus.  It  is  the  cause  of  pneumonia  in  about 
7  per  cent  of  the  cases.  It  is  taken  as  the  type  of  a  group, 
"  the  mucosus  capsulatus  "  group,  and  is  also  called  B. 
mucosus  capsulatus. 


NON-SPORING    BACILLI 


241 


Characteristics. — A  short  plump  bacillus  with  rounded 
ends,  but  showing  some  very  long  forms  (o-6  to  5  micra  long 
by  0-5  to  i;5  micron).  The  short  thick  forms  are  mostly 
found  in  animal  tissues,  and  at  times  are  almost  coccoid. 
In  sputum  it  shows  a  capsule,  and  in  other  preparations 
from  the  body.  It  occurs  in  pairs,  and  hence  has  been 
called  a  diplobacillus.  It  may  also  form  short  chains. 
It  is  non-motile,  non-flagellar,  non-sporing,  non-gelatin- 
liquefying,  and  non-Gram  (i.e.,  Gram-negative). 

Cultures. — It  grows  readily  on  ordinary  media  and  in 
gelatin  at  room  temperature.  It  grows  well  on  acid  or 
alkaline  media,  is  aerobic,  and  facultatively  anaerobic. 

In  broth  :  rapid  abundant  growth  with  a  pellicle,  general 
clouding,  and  later  a  stringy  sediment. 

On  agar  :  sticky  mucus-like  colonies  of  a  greyish-white 
colour. 

In  gelatin  stab  :  a  white  line  of  growth  at  first,  but  with 
increasing  growth  at  surface  a  '  nail-head  "  appearance 
is  produced.  This  was  at  one  time  thought  to  be  peculiar 
to  Friedlaender's  bacillus. 

On  potato  :    abundant,  somewhat  brownish  growth. 

In  peptone  water  :   no  indol  formation. 

In  milk :  abundant  growth  with  capsule  formation. 
Acid  and  clot  are  slowly  formed. 

Pathogenicity. — For  man  :  pneumonia  of  a  severe  and 
fatal  type  ;  ulcerative  stomatitis  and  nasal  catarrh ; 
acute  tonsillitis ;  in  antral  suppurations  and  in  foetid 
coryza  ;   and,  on  rare  occasions,  in  septicaemia. 

For  animals  :  a  mouse  injected  at  the  root  of  the  tail 
dies  in  two  days  of  septicaemia.  It  is  also  pathogenic  for 
guinea-pigs  ;   less  so  for  rabbits. 


Distinction  from 

THE    PNEUMOCOCCUS. 

Pneumococcus 

Pneumobacillus 

Growths  on  ordinary  media 
on  gelatin 
, ,        in  milk 
Staining 

Sparse 

Almost  none     .  . 
Acid  +  clot 
Gram-positive .  . 

Good 

Nail-head  growth 
Acid  +  clot  (late) 
Gram-negative 

Allied   bacilli   are :   B.    ozaenae,    found    in    foetid   nasal 
catarrh,    which   is   scarcely   separable   from   B.    mucosus, 

16 


242         PUBLIC    HEALTH    BACTERIOLOGY 

capsulatus ;  bacillus  of  rhinoscleroma.  Both  these 
bacilli  differ  only  in  not  fermenting  dextrose. 

B.  Lactis  Aerogenes — Is  a  widely  distributed  organism, 
and  was  isolated  by  Escherich  in  1885  from  the  faeces  of 
infants.  It  is  almost  constantly  present  in  milk,  faeces, 
sewage,  and  water.  It  differs  from  B.  coli,  which  is  found 
in  like  circumstances,  in  being  non-motile  and  non- 
flagellar,  in  possessing  a  capsule  in  milk  cultures,  in 
fermenting  saccharose  and  starch  but  not  dulcite  ;  and 
in  not  forming  indol. 

Cultures. — It  grows  readily  on  all  media. 

In  broth,  it  forms  a  pellicle,  and  causes  general  clouding. 

On  agar  and  gelatin,  it  forms  a  heavy  white  growth. 

In  gelatin  stab,  it  gives  a  nail-head  growth. 

On  potato,  it  grows  well  and  forms  gas  from  the  starch. 

In  Milk,  acid  formation  and  coagulation  are  rapid.  The 
clot  is  not  digested  by  the  bacillus,  and  in  ordinary  souring 
of  milk  the  germs  present  which  produce  proteolytic 
ferments  have  their  growth  restrained  by  the  large  amount 
of  lactic  acid  formed  by  the  more  rapid  action  of  B. 
lactis  aerogenes.  It  is  scarcely  pathogenic,  though 
flatulence  in  infants  has  been  attributed  to  its  action, 
and  a  cystitis  in  which  gas  was  formed  in  the  bladder, 
associated  with  an  acid  urine.  For  animals,  its  patho- 
genicity is  not  property  established,  the  reports  being 
contradictory.  It  is  an  aerobe,  but  a  facultative  anaerobe. 
Optimum  temperature,  250  to  300  C.  B.  lactis  aerogenes 
is  distinguished  from  Friedlaender's  bacillus  by  its  invari- 
able and  rapid  curdling  of  milk;  but  some  authorities 
consider  it  to  be  identical  with  that  organism. 


BACILLUS     ACIDI     LACTICI     (HUEPPE), 

This  bacillus  is  present  in  milk,  which  it  curdles  and 
acidifies.     It  does  not  ferment  saccharose  or  dulcite. 

B.  acidi  lactici  (Leichmann)  is  believed  to  be  really  a 
streptococcus,  the  Streptococcus  lacticus  (Kruse),  which 
Heinemann  states  is  a  variety  of  the  Streptococcus  pyo- 
genes. It  is  present  on  the  cow's  hide,  in  cow  dung,  and 
in  milk  from  the  first  stage  of  milking. 


NON-SPORING    BACILLI  243 

MINUTE     BACILLI. 

Under  this  heading  may  be  conveniently  grouped  the 
bacilli,  of  influenza,  of  acute  epidemic  conjunctivitis,  and 
of  whooping-cough.     All  are  Gram-negative. 

Bacillus  Influenzae — Was  first  described  in  1892  by 
several  observers,  and  has  been  called  after  one  of  them 
the  Pfeiffer  bacillus.  It  is  very  small  even  among  micro- 
organisms, being  only  from  0-5  to  1-2  micron  long  by  0-2  to 
0-4  micron  thick.  A  tubercle  bacillus,  3  micra  by  0-3 
micron  is  thus  equal  in  length  to  several  (3  to  6)  influenza 
bacilli  placed  end  to  end. 

It  is  then  a  small  bacillus,  of  irregular  length,  having 
rounded  ends,  rarely  forming  chains,  non-motile,  non- 
sporing,  Gram-negative,  and  not  growing  on  gelatin  or  at 
room  temperatures.  It  is  not  easily  stained  with  the  usual 
dyes  ;  best  with  10  per  cent  aqueous  fuchsin,  or  Loerfler's 
methylene-blue,  5  minutes  of  either.  The  bacilli  form 
irregular  clusters  ;  occasionally  polar  staining  is  noticed. 

Cultures. — It  is  not  easily  cultivated,  growing  only  in 
the  presence  of  haemoglobin.  This  is  obtained  on  the 
ordinary  media  by  smearing  them  with  some  blood  drawn 
from  the  finger,  or  by  mixing  melted  agar  with  fresh  blood. 
The  blood  of  the  pigeon  may  be  used.  The  medium  is 
inoculated  with  the  sputum  coughed  up  from  the  bronchi, 
avoiding  mucus  from  the  mouth.  Colonies  appear  in  18 
to  24  hours,  as  minute  transparent  drops,  colourless,  and 
likened  to  drops  of  dew.  Growth  ceases  in  2  to  3  days. 
Frequent  subculturing  and  storage  at  room  temperature 
are  needed  to  keep  the  cultures  alive. 

It  is  aerobic,  and  shows  no  growth  under  strict  anaero- 
biosis.  It  is  readily  killed  at  6o°  C.  ;  and  by  drying,  in 
a  few  hours.  Dies  in  culture  media  within  a  week.  Its 
usual  habitat  during  an  epidemic  is  the  nasal  passages 
and  bronchial  tubes.  It  is  said  to  remain  in  these 
places  after  recovery  from  the  attack,  and  to  persist  for 
years.  The  immunity  produced  by  an  attack  of  influenza 
is  very*  short.  A  pseudo-bacillus,  differing  only  in  its 
slightly  larger  size  and  its  growth  in  threads,  and  showing 
involution  forms,  has  been  described,  but  its  differentiation 
is  doubtful. 


244  PUBLIC     HEALTH     BACTERIOLOGY 

Koch-Weeks  Bacillus. — A  bacillus  similar  to  the 
above,  but  longer  and  more  slender,  was  described  by- 
Koch  in  1883,  and  by  Weeks  in  1887,  in  connection  with  an 
epidemic  form  of  acute  conjunctivitis. 

Cultures. — It  grows  best  on  serum  agar,  and  at  370  C ; 
the  colonies  appear  in  36  hours  as  dew  drops. 

The  disease  is  characterized  by  a  muco-purulent  discharge, 
hyperaemia  of  the  whole  of  the  conjunctiva,  and  swelling 
of  the  lymph  follicles  of  the  lids,  which  show  through 
the  palpebral  conjunctiva  as  slightly  raised  pinkish-grey 
bodies,  a  half  to  one  millimetre  in  diameter.  A  film  made 
from  the  discharge  and  stained  with  Loeffler's  methylene- 
blue,  shows  the  bacilli.  The  affection  is  very  contagious, 
and  to  prevent  epidemics  in  schools,  common  face-towels 
should  be  rigorously  prohibited. 

Bordet-Gengou  Bacillus.  —  These  observers  found 
a  small  ovoid  bacillus  in  the  sputum  of  a  child  suffering 
from  whooping-cough. 

Culture. — In  1906,  six  years  later,  they  succeeded  in 
cultivating  it  on  a  special  medium,  after  failing  with 
ascitic  agar  and  blood  agar.  This  medium  is  a  glycerin 
extract  of  potato  with  4  per  cent  salt  and  2-5  per  cent 
agar,  to  which  is  added  an  equal  quantity  of  defibrinated 
human  or  rabbit's  blood.  On  this,  inoculated  from 
sputum,  the  colonies  appear  within  48  hours,  and  are  small, 
greyish,  and  rather  thick.  In  subcultures,  they  give 
a  more  luxuriant  growth,  and  can  then  be  grown  on  blood 
agar  and  in  ascitic  broth,  in  which  it  causes  a  viscid 
sediment  but  no  pellicle.  It  is  strictly  aerobic,  and  grows 
moderately  below  blood-heat.  It  remains  alive  in  culture  for 
as  long  as  two  months.  Specific  agglutinins  are  developed 
in  immunized  animals,  which  serve  to  distinguish  it  from 
B.  influenzae.  The  washed  and  dried  bacilli  ground  in  a 
mortar  and  injected  into  a  rabbit  intravenously,  usually 
kill  it  in  24  hours.  Specific  complement  fixation  has 
been  used  by  Bordet  and  Gengou  to  prove  the  identity  of 
the  bacillus,  using  the  serum  of  an  infant  suffering  from 
whooping-cough. 

The  organism  is  present  in  the  sputum  in  the  early 
stages  in  predominating  numbers,  but  later  it  is 
swamped  by  others.     It  is  scattered  among  the  pus  cells, 


NON-SPORING    BACILLI  245 

and  at  times  is  intracellular.  It  is  extremely  small  and 
ovoid,  so  that  it  is  readily  mistaken  for  a  micrococcus. 
It  is  slightly  larger  than  the  bacillus  of  influenza,  and 
more  ovoid.  It  is  Gram-negative,  stains  with  Loeffler's 
methylene-blue,  dilute  carbol-fuchsin,  or  aqueous  fuchsin. 
Toluidin  blue  in  a  special  medium  is  advised.  (Toluidin 
blue  5  grm.,  alcohol  ioo  c.c,  water  500  c.c.  ;  dissolve  ; 
add  of  5  per  cent  aqueous  carbolic  acid  solution,  50  c.c.  ; 
stand  two  days  ;   filter.) 

Bacillus  of  Ducrev — Is  a  small  bacillus  (1  to  2  micra 
X  0-5  micron),  regularly  found  in  soft  chancre  or  chancroid. 
It  is  non-motile,  non-flagellar,  non-sporing,  Gram-negative, 
and  is  found  at  times  inside  the  leucocytes. 

Culture. — It  only  grows  on  whole  blood  agar,  and  dies 
off  at  room  temperatures.  Colonies  show  in  about  48 
hours.  Inoculation  of  pure  cultures  on  the  skin  produces 
typical  chancres  in  4  to  6  days. 

Zur  Nedden's  Bacillus. — A  small  slightly  curved 
bacillus  (1  micron  long)  which  has  been  isolated  in  ulcera- 
tive conditions  of  the  cornea.  It  grows  well  on  the 
ordinary  media.  It  is  non-motile,  Gram-negative,  and 
does  not  liquefy  gelatin.  It  clots  milk,  forms  acid  with 
glucose,  but  no  indol  in  peptone  water. 

Culture. — It  grows  well  on  potato. 

MORAX-AXENFELD     DIPLO-BACILLUS. 

This  is  a  short,  thick  bacillus  with  rounded  ends,  found 
in  a  chronic  eye  condition  called  "  angular  conjunctivitis," 
in  which  there  is  slight  redness  of  the  edges  of  the  lids, 
especially  at  the  angles.  The  ocular  conjunctiva  is  seldom 
affected.  It  mainly  affects  adults,  and  chiefly  women. 
There  is  rarely  any  corneal  trouble.  It  was  described  by 
Morax  in  1896,  and  by  Axenfeld  a  year  later.  The  diag- 
nosis is  easily  made,  by  taking  a  smear  of  the  small  bead 
of  pus  which  gathers  through  the  night  at  the  angles,  and 
staining  with  the  usual  dyes.  The  bacillus  is  easily  stained, 
and  is  Gram-negative.  In  stained  films,  short  and  long 
chains  of  polar  stained  bacilli  are  seen. 

Culture. — It  grows  best  on  Loeffler's  blood  serum  at 
370  C,  causing  small  pits  of  liquefaction  in  48  hours. 


246         PUBLIC    HEALTH    BACTERIOLOGY 
DIPHTHERIA     BACILLUS. 

Klebs  first  described  the  bacillus  in  the  throat  mem- 
brane in  1883,  and  Loeffler  first  cultivated  it  in  1884  ; 
hence  called  the  Klebs-Loeffler  bacillus.  The  toxins  pro- 
duced by  it  were  investigated  by  Roux  and  Yersin  in  1888- 
89,  and  the  antitoxins  by  Behring  and  Kitasato  in  1890. 

Description. — A  slender  bacillus,  1  to  6  micra  long  and 
0-3  to  i-i  micron  broad.  From  the  throat  they  are  mostly 
4  to  5  micra  long.  They  are  rarely  of  uniform  thickness 
throughout,  showing  club-shaped  thickening  at  one  or  both 
ends.  They  are  straight  or  slightly  curved,  stain  deeply 
with  methylene-blue,  often  showing  granules  more  darkly 
stained,  so  that  a  dotted,  beaded,  or  striped  or  barred 
appearance  results.  The  longer  individuals  often  have 
a  strong  resemblance  to  short  chains  of  streptococci.  In 
18-hour-old  cultures,  many  of  the  bacilli  show  on  staining 
deeply  stained  oval  bodies  situated  most  frequently  at  the 
ends,  the  so-called  "  polar  "  or  "  Babes-Ernst  "  bodies. 
These  were  first  regarded  as  spores,  but  are  now  considered 
to  be  chromatic  granules.  B.  diphtheriae  is  an  aerobe.  Is 
non-motile,  non-sporing,  non-flagellar,  and  non-liquefying 
of  gelatin.     It  is  Gram-positive. 

Cultures. — Grows  on  all  media,  but  quickest  and  most 
characteristically  on  Loeffler' s  blood  serum  :  [beef-blood 
serum,  3  parts,  1  per  cent  glucose  broth  (meat  infusion), 
1  part.  Put  in  tubes,  slant,  coagulate  at  700  C,  and 
sterilize  at  570  C.  for  1  hour  on  eight  days.]  On  this 
medium  colonies  form  in  from  12  to  24  hours  at  370  C,  as 
small  circular  discs  of  opaque  whitish  colour,  like  candle- 
grease  spots,  and  enlarging  rapidly,  outstrip  any  accom- 
panying colonies  of  streptococci. 

On  agar,  a  similar  growth  occurs,  but  less  quickly,  and 
is  closely  resembled  by  that  of  Streptococcus  pyogenes. 

In  broth,  a  pellicle  may  form,  and  a  turbidity  which 
however  soon  settles  to  the  bottom,  leaving  the  fluid  clear 
and  producing  a  powdery  deposit. 

It  grows  well  in  milk  but  does  not  clot  it. 

Does  not  form  indol.  Ferments  glucose,  galactose, 
laevulose,  maltose,  and  glycerin,  but  not  mannite  or 
saccharose.     It  reduces  nitrate  to  nitrite. 


NON-SPORING    BACILLI  247 

Grows  best  at  370  C.  ;  growth  at  220  C,  but  not  at 
20°  C. 

Isolation. — Take  sterile  swab,  rub  on  throat,  then  rub  over 
serum  in  tube,  and  incubate  for  18  to  24  hours  at  370  C. 
Take  platinum  loop  and  rake  all  over  surface  of  serum, 
and  then  make  a  film  and  stain  with  Loemer's  methylene- 
blue  or  Neisser.  Distinguish  from  Hoffmann's  bacillus, 
which  is  shorter  and  plumper,  does  not  produce  acid  in 
glucose,  is  non-pathogenic  to  guinea-pigs,  and  does  not  show 
granules  with  Neisser.  (B.  diphtherias  at  times  does  not 
give  Neisser,  and  occasionally  pseudo-B.  diphtherias  does 
stain  with  Neisser.)  The  only  sure  method  of  differentia- 
tion is  toxicity  of  culture ;  guinea-pig  killed  in  24  to  48  to 
72  hours  by  injection  of  toxin  of  true  B.  diphtherias. 

In  the  serum-water  medium  of  Hiss,  plus  1  per  cent  of 
glucose,  lasvulose,  galactose,  maltose,  lactose,  saccharose, 
mannite  and  dextrin,  B.  diphtherias  produces  acid  in  all 
except  with  mannite,  lactose,  and  saccharose  ;  B.  xerosis 
produces  acid  with  all  except  mannite,  lactose,  and  dextrin  ; 
B.  Hoffmanni  does  not  produce  acid  with  any. 

The  saccharose  and  dextrin  media  therefore  serve  to 
differentiate  :  B.  diphtheria,  acid  with  dextrin,  not  with 
saccharose  ;  B.  xerosis,  acid  with  saccharose,  not  with 
dextrin  ;   B.  Hoffmanni,  acid  with  neither. 

In  Vincent's  angina,  B.  fusiformis  is  found  :  an  anaerobe, 
Gram-negative,  longish,  and  swollen  in  the  middle. 

B.  xerosis  is  found  in  the  conjunctiva,  and  is  pfactically 
identical  with  the  B.  diphtherias :  it  is  non-pathogenic  to 
animals ;  produces  no  toxin  ;  does  not  form  acid  with 
glucose,  but  ferments  saccharose. 

Habitat. — Mucous  surfaces,  mouth,  throat,  nose,  con- 
junctiva, larynx,  middle  ear,  vagina. 

Thermal  Death-Point. — Forty-five  minutes  at  550  C, 
(moist  heat)  ;  dry  membrane,  1  hour  at  980  C. 

Resistance. — Lives  six  to  eight  weeks  on  agar,  five  to  six 
months  on  serum,  twelve  to  fifteen  months  on  dextrose 
serum.     In  dried  membrane  retains  vitality  for  months. 

Pathogenicity. — Guinea-pigs  die  in  from  two  to  three 
days  after  subcutaneous  injection  of  young  broth  culture. 
Nephritis  and  paralysis  are  observed,  but  the  characteristic 
feature   is   enlarged   and   hasmorrhagic   condition    of   the 


248  PUBLIC    HEALTH    BACTERIOLOGY 

adrenals.  Cats,  dogs,  and  pigeons  are  very  susceptible  to 
mucus  infection  ;   rats  and  mice  are  refractory. 

A  guinea-pig  killed  by  the  injection  of  a  virulent  culture 
of  diphtheria  bacillus  shows  congestion  of  all  the  organs, 
especially  severe  in  the  suprarenals. 

Toxins. — Diphtheria  toxin  is  an  extracellular  one,  that 
is,  it  is  soluble  in  the  liquid  media,  and  can  be  obtained 
separate  from  the  bacillus  by  filtration  through  a  Chamber- 
land  tube.  Toxin  formation  is  best  got  in  meat-infusion 
broth  with  added  peptones,  and  rendered  alkaline,  after 
two  to  three  weeks'  growth  at  37-5°  C.  A  free  supply  of 
oxygen  is  important  to  secure  the  greatest  toxin  formation. 

The  toxin  is  readily  destroyed  by  bright  light,  by 
exposure  in  a  liquid  solution  to  6o°  C,  or  if  in  dry  state 
to  a  temperature  over  700  C.  Sealed  and  kept  in  the  dark 
and  in  the  cold,  it  may  be  kept  for  long  periods.  It  is 
believed  to  be  closely  allied  to  the  albumoses. 

Antitoxin. — The  mode  of  preparation  and  standardiza- 
tion is  described  under  Antitoxic  Sera  (page  189).  The 
value  of  antitoxin  in  the  treatment  of  diphtheria  is  now 
well  attested.  The  results  are  better  the  earlier  the  injec- 
tion, so  that  few  deaths  occur  in  those  injected  within 
twenty-four  hours  of  the  onset,  the  rate  gradually  rising 
until  by  the  fifth  day  the  effect  is  slight.  The  dose 
given  is  not  proportionate  to  the  age,  but  to  the  severity 
of  the  attack  and  the  time  that  has  elapsed  before 
coming  under  treatment.  2000  to  8000  units  should  be 
given  to  children  on  these  principles,  and  up  to  50,000 
units  have  been  given  in  one  case.  In  large  doses,  it  is 
better  to  give  the  higher-potency  sera,  in  which  10,000 
units  can  be  had  in  10  c.c.  of  serum.  In  about  one-third 
of  the  cases  injected,  serum  sickness  or  serum  disease  is 
noted.  The  symptoms  are  :  an  erythematous  rash  and 
fever  coming  on  in  a  week  to  ten  days  after  the  injection. 
At  times  general  pains  and  even  arthritis  may  be  present. 
These  symptoms  are  more  likely  to  be  produced  by  large 
doses  of  serum.  But  more  alarming  symptoms  than  these 
occur  soon  after  the  injection,  as  has  been  referred  to 
under  Anaphylaxis  (page  212).  On  analysis  these  cases 
are  found  to  occur  in  people  who  have  not  been  previously 
injected,  and  in  people  who  have  had  a  previous  injection 


NON-SPORING    BACILLI  249 

more  than  the  incubation  period  of  the  serum  disease 
(that  is,  ten  to  twelve  days)  before.  On  account  of  these 
facts,  Goodall  advises  that  prophylactic  doses  of  diphtheria 
antitoxin  should  not  be  given  to  anyone  without  discrimina- 
tion, and  never  to  a  person  the  subject  of  asthma  (see 
page  213) .  In  an  actual  attack  of  diphtheria  in  such  a  person 
the  risk  would  have  to  be  definitely  considered,  and  a 
judgment  come  to  on  the  relative  dangers  of  the  attack 
and  the  use  of  the  antitoxin.  In  those  who  have  had  a 
previous  dose,  either  for  an  attack  or  for  prophylaxis,  some 
time  antecedent,  one  should  be  on  the  outlook  for  alarming 
symptoms  if  a  second  dose  is  being  given.  It  is  stated 
that  so  far  no  death  has  been  recorded  in  these 
circumstances,  which  is  to  some  extent  reassuring.  This 
class  of  case  suggests  the  avoidance  of  prophylactic  doses 
altogether,  as  if  the  person  takes  an  attack  more  than 
ten  days  later,  he  is  sensibilized  to  the  now  required 
injection.  (See  for  further  details,  articles  by  Goodall  in 
Public  Health,  January,  191 1,  and  in  Encyclopedia  Medica  ; 
and  by  Currie,  Journal  of  Hygiene,  January,  1907.  Also 
annotation  in  Lancet,  1911,  vol.  i,  page  1654.) 

Antitoxic  serum  keeps  well  in  a  cool,  dark  place. 
Anderson  has  found  that  at  200  C.  the  average  yearly  loss 
of  potency  is  20  per  cent  ;  at  150  C,  it  is  about  10  per  cent  ; 
and  at  50  C,  it  is  only  about  6  per  cent.  Dried,  and  kept 
at  50  C,  its  potency  was  practically  unimpaired  after  5-5 
years.  The  addition  of  chloroform,  tricresol,  etc.,  to 
preserve,  had  no  influence  apparently  on  the  rate  of 
deterioration. 


250  PUBLIC    HEALTH     BACTERIOLOGY 

BACILLUS     MALLEI. 

The  B.  mallei  is  the  bacillus  of  glanders,  a  disease  of 
horses,  mules,  and  asses.  Horned  cattle  are  quite  immune, 
whilst  goats  and  sheep  are  intermediate  in  susceptibility. 
Guinea-pigs  and  rabbits  can  be  infected  by  inoculation,  but 
rats  are  immune. 

It  was  first  obtained  in  pure  culture  and  accurately 
studied  by  Loeffler  and  Schuetz  in  1882,  and  from  the 
human  subject  by  Weichselbaum  in  1885. 

Description. — B.  mallei  is  a  medium-sized  rod  (3  to  4 
micra  X  0-5  to  0-75  micron),  straight  or  slightly  curved, 
usually  with  rounded  ends.  It  is  about  the  same  length  as 
the  tubercle  bacillus,  but  distinctly  thicker.  These  bacilli 
show  considerable  variations  in  size,  even  in  the  same 
culture  ;  and  this  is  characteristic.  They  are  non-motile, 
non-flagellar,  non-sporing,  and  Gram-negative.  They 
usually  appear  as  single  bacilli ;  on  rare  occasions  short 
filamentous  forms  occur. 

Staining. — The  bacilli  stain  rather  easily,  but  are  as  easily 
decolorized.  The  best  results  are  got  by  staining  with  a 
mordant  present,  and  simply  washing  in  water,  and 
drying  ;  in  the  case  of  tissues,  dehydrating  by  the  aniline- 
oil  method.  Carbol-fuchsin  and  carbol-thionin-blue  make 
good  stains.  Loefner's  methylene-blue,  followed  by  slight 
decolorization  in  weak  acetic  acid,  and  then  fifteen  minutes 
in  saturated  solution  of  tannic  acid,  wash,  dry,  and  mount, 
gives  good  results.  As  a  counter-stain,  1  per  cent  acid 
fuchsin  for  half  a  minute  may  be  used.  With  methylene- 
blue,  the  bacilli  stain  irregularly ;  granular,  deeply  staining 
areas  alternating  with  unstained  or  faintly-stained  portions. 
This  has  been  ascribed  to  degeneration,  and  to  preparation 
towards  spore  formation.  It  is  probably  a  peculiarity  of 
the  cell  protoplasm. 

Cultures. — Grows  well  on  all  media  at  350  to  370  C., 
indifferent  to  moderate  degrees  of  acid  or  alkali.  Glycerin 
or  glucose  render  the  media  even  more  favourable. 

On  agar  and  on  glycerin-agar  :  the  growth  is  greyish 
white  to  yellow. 

On  gelatin  :  growth  is  slow,  and  greyish-white  ;  no  lique- 
faction takes  place. 


NON-SPORING    BACILLI  251 

In  broth :  diffuse  clouding ;  later,  a  heavy,  tough,  slimy 
sediment  forms,  and  the  broth  becomes  brown. 

In  milk  :  coagulation  takes  place  slowly,  with  slight  acid 
formation. 

On  potato  :  the  growth  at  37 °  C.  is  characteristic.  Growth 
is  rapid  and  abundant,  and  in  forty-eight  hours  forms  a 
transparent  layer  over  the  whole  surface,  and  of  a  yellowish 
tint,  like  clear  honey.  The  growth  gets  darker  and  more 
opaque,  and  on  the  eighth  day  it  is  reddish-brown  or 
chocolate  in  colour,  and  at  the  edge  the  potato  is  stained 
a  greenish-yellow  colour.  Spirilla  cholerae  and  Metchni- 
kovi,  and  B.  pyocyaneus  give  similar  cultures. 

Resistance. — Killed  by  one  hour  at  750  C.  or  two  hours 
at  6o°  C.  In  the  dark,  in  sealed  tubes  and  on  artificial 
media,  it  may  remain  alive  for  years.  In  watering-troughs, 
it  has  been  found  after  seventy  days.  Complete  drying 
kills*  it  in  a  short  time ;  carbolic  1  per  cent,  in  30  min. ; 
corrosive  sublimate  o-i  per  cent,  in  15  min. 

Pathogenicity. — Notable  for  horses,  mules,  asses,  cats, 
dogs,  guinea-pigs,  rabbits,  and  field  mice.  Non-sus- 
ceptible animals  are  :  cattle,  pigs,  birds,  rats,  house  mice, 
and  white  mice.  In  the  horse,  the  disease  takes  two  forms. 
When  affecting  the  superficial  lymphatic  glands,  it  is  called 
"farcy  ;  "  when  affecting  the  nasal  mucous  membrane  it 
is  called  "  glanders,"  and  is  a  much  more  serious  disease. 
In  glanders  the  course  may  be  acute  or  chronic.  In  the 
acute-  form,  chill  is  followed  by  general  high  temperature, 
and  in  a  few  days  the  nasal  mucous  membrane  is  studded 
with  nodules,  there  is  profuse  nasal  discharge,  and  later, 
ulceration  of  the  nodules  and  swelling  of  the  corresponding 
glands,  and  these  also  tend  to  break  down.  Finally  the 
lungs  are  involved,  and  death  takes  place  in  from  one  to 
four  weeks.  In  the  chronic  form,  the  onset  is  more  gradual, 
the  nasal  swelling  being  accompanied  by  subcutaneous 
swellings  all  over  the  body,  some  of  which  tend  to  break 
down  and  ulcerate.  These  swellings  are  the  so-called 
"  farcy  buds,"  and  may  persist  for  years. 

In  man,  the  onset  is  usually  violent,  w7ith  fever  and 
general  symptoms;  and  most  cases  terminate  fatally 
within  two  to  three  weeks,  sometimes  within  a  few  days. 
The  infection  is  usually  by  a  wound,  which  is  followed  by 


252  PUBLIC    HEALTH    BACTERIOLOGY 

lymphangitis,  and  then  a  general  infection  resembling  a 
pyaemia.  As  in  the  horse,  the  nasal  mucosa  tends  to 
become  affected.     At  times  the  course  is  more  chronic. 

In  the  horse,  the  infection  is  usually  by  the  mucous 
membrane  of  the  nose  or  mouth,  by  wounds,  or  at  times 
by  the  alimentary  canal. 

Toxin. — No  soluble  toxin  is  described,  but  a  concentrated 
three-weeks'  culture  in  glycerin  broth,  sterilized  by  heat 
and  filtered,  is  used  as  a  diagnostic  agent  under  the  name 
of  mallein  (fluid).  Dry  mallein  has  been  prepared  by 
filtering  a  broth  culture,  concentrating  filtrate  on  a  water- 
bath  to  one-tenth  of  its  bulk,  and  precipitating  with 
thirty  times  its  bulk  of  alcohol.  Mallein  differs  from  many 
other  bacterial  extracts  in  being  extremely  resistant  to 
heat  and  storage  without  loss  of  strength  ;  1200  C.  has  no 
destructive  effect  on  it. 

Diagnosis  of  Glanders. — Three  tests  are  used,  namely  : 
(i)  Guinea-pig  inoculation  ;  (2)  Mallein  test  ;  (3)  Agglu- 
tination test. 

1.  Inoculation  of  a  Guinea-pig. — A  male  guinea-pig  is 
injected  intraperitoneally  with  fragments  of  the  diseased 
tissue,  scrapings  from  ulcers,  or  nasal  discharge  of  the 
suspected  animal. 

A  positive  reaction  is  shown  by  the  testicles  becom- 
ing red  and  swollen  usually  on  the  second  or  third  day, 
due  to  inflammation  of  the  tunica  vaginalis.  Severe 
general  symptoms  follow,  and  death  occurs  in  twelve  to 
fifteen  days.  Greyish  nodules  are  found  in  the  spleen  and 
other  organs.  The  test  is  not  absolutely  specific,  but  is 
useful  when  other  tests  are  inapplicable.  A  culture  on 
potato  of  the  pus  from  the  tunica  vaginalis  should  be 
made. 

2.  Mallein  Test. — A  proper  dose  of  mallein  is  infected 
subcutaneously  into  the  breast  or  neck  of  the  suspected 
animal.  It  is  advised  to  inject  a  dose  into  a  control 
animal.  The  temperature  of  the  animal  should  be  taken 
at  least  three  times  a  day  for  one  or  two  days  before 
injection.  The  injection  is  made  at  6.0  to  7.0  a.m.,  and 
the  reaction  will  be  at  its  height  at  or  before  10  p.m.  of  the 
same  day.  The  temperature  is  taken  every  two  hours 
after  the  injection  for  at  least  eighteen  hours.      On  the 


NON-SPORING    BACILLI  253 

succeeding  day  take  the  temperature  at  least  three  times. 
In  a  healthy  animal  free  from  glanders,  a  local  swelling,  not 
exceeding  3  inches  in  diameter,  is  produced  at  the  seat 
of  inoculation,  and  a  rise  of  temperature  not  exceeding 
i°  C.  (i-8°  F.)  ;  and  both  swelling  and  temperature  have 
much  subsided  in  twenty-four  hours.  In  a  horse  suffering 
from  glanders,  there  appears  within  a  few  hours  a  firm,  hot, 
diffuse  swelling,  which  reaches  a  maximum  size  in  twenty- 
four  hours,  is  intensely  tender  during  that  time,  and  lasts 
from  three  to  nine  days.  The  size  of  the  swelling  reaches  at 
least  5  inches  in  diameter.  The  temperature  rises  in  six  to 
eight  hours  1-5°  to  2°  C.  (270  to  3-6°  F.),  reaching  1040  to 
1060  F.  The  high  temperature  continues  for  eight  to  ten 
hours  (maximum  about  ten  to  sixteen  hours  after 
injection),  and  then  gradually  falls,  but  is  distinctly  above 
normal   on  the  following  day.     This  reaction  is  specific. 

3.  Agglutination  Test. — The  macroscopic  or  sedimentation 
method  in  high  dilutions  (1-1000)  is  preferred.  Normal 
horse  serum  may  react  in  1-500. 

Immunity. — An  attack  of  glanders  does  not  confer 
immunity.  Artificial  active  immunization  has  been 
attempted  but  has  so  far  failed. 

Nodules. — The  nodules  found  in  glanders  show  more 
leucocytic  infiltration  and  less  proliferative  change  towards 
formation  of  epitheloid  cells  than  does  tubercle. 

Prevention. — The  Glanders  or  Faro*  Order  of  1907, 
issued  by  the  Board  of  Agriculture,  (i)(jLays  down  com- 
pulsory notification  of  actual  or  suspected  disease  ; 
(2)  Empowers  local  authority  to  slaughter  at  once  any 
diseased  horse,  ass,  or  mule  ;  (3)  Enables  local  authority 
to  test  suspected  animals  with  mallein,  and  deal  with 
contacts. 


25i         PUBLIC    HEALTH    BACTERIOLOGY 

BACILLUS     PESTIS. 

This  plague  bacillus  belongs  to  a  group  of  bacilli  which 
are  all  highly  pathogenic  to  the  animal  world,  and  produce 
in  them  a  haemorrhagic  septicaemia.  The  bacilli  now 
usually  classed  in  this  group  are  :  the  bacillus  of  human 
plague,  of  swine  plague,  of  chicken  cholera,  of  septic  pleuro- 
pneumonia in  cattle,  and  of  rabbit  septicaemia.  The  group 
characteristics  are  :  short,  plump,  non-motile  bacilli ;  non- 
flagellar  ;  non-sporing  ;  Gram-negative  ;  non-gelatin-liquefy- 
ing ;  strongly  aerobic,  growing  readily  on  simple  media, 
easily  stained  but  showing  a  marked  tendency  to  stain 
more  deeply  at  the  ends  than  at  the  centre  (bipolar 
staining).  They  are  believed  by  some  to  be  varieties  of 
one  organism. 

Plague  is  a  specific,  infective  disease,  caused  by 
the  B.  pestis,  and  characterized  by  inflammation  of  the 
lymphatic  glands  (buboes),  carbuncles,  pneumonia,  and 
often  haemorrhages  (Osier).  In  the  past  the  plague  has 
occurred  in  tremendous  epidemics,  and  even  to-day  in 
India  it  proves  a  terrible  scourge.  The  large  and  extremely 
fatal  epidemic  of  pneumonic  plague  in  China  in  1910-11 
shows  that  it  still  has  very  pathogenic  powers  for  mankind. 

In  the  sixth  century,  in  the  reign  of  Justinian,  Emperor 
of  Rome,  half  the  population  of  the  Roman  Empire 
perished  of  the  disease.  In  the  fourteenth  century 
the  "  black  death "  overran  Europe  and  destroyed 
25,000,000,  or  about  one-fourth  of  the  population.  In  the 
seventeenth  century  it  raged  virulently,  and  in  London 
alone,  in  1665,  about  70,000  people  died.  During  the 
eighteenth  and  nineteenth  centuries  its  ravages  lessened. 

In  1893  an  outbreak  appeared  at  Hong-Kong,  and  since 
then  the  disease  has  occurred  in  many  parts  of  the  world, 
notably  in  India  since  1896,  in  Egypt,  in  South  Africa, 
and  in  several  Mediterranean  ports,  and,  after  an  absence 
from  Great  Britain  of  over  two  hundred  years,  it 
obtained  a  foothold  in  Glasgow  in  1900.  It  reached  New 
York  quarantine  station  in  1899,  and  in  San  Francisco 
broke  out  in  1900,  continuing  until  1904.  In  Australia, 
cases  appeared  at  Sydney  and  other  ports.  In  the  county 
of  Suffolk,  in  England,  an  epidemic  of  rat  plague,  associated 


NON-SPORING    BACILLI  255 

with  a  limited  outbreak  of  pneumonic  plague  in  man,  was 
(September,  1910)  the  occasion  of  considerable  anxiety  and 
of  increased  vigilance  and  action.  In  California  the  last 
case  of  plague  was  noted  in  1909,  but  an  epizootic  of  plague 
among  squirrels,  causing  thousands  of  deaths  among  these 
animals,  was  only  reported  as  quiescing  in  191 1. 

In  1894,  Kitasato  and  Yersin  independently  discovered 
the  bacillus  in  large  numbers  in  the  buboes,  cultivated  it 
in  pure  growth,  and  reproduced  the  disease  in  susceptible 
animals  by  inoculation,  and  from  them  recovered  the 
bacillus.  The  proof  in  the  human  subject  was  given 
later,  when  by  an  accidental  infection  a  physician  and  a 
nurse  died  of  plague. 

Description. — B.  pestis  is  a  short  thick  bacillus  with 
well-rounded  ends,  thus  appearing  as  a  small  oval  rod, 
two  to  three  times  in  length  what  it  is  in  breadth  (1-5 
to  175  X  0-5  to  07).  The  bacilli  appear  singly,  though 
at  times  in  pairs,  and  in  fluid  cultures  in  chains.  In 
young  cultures  they  show  marked  variations  in  size,  and 
less  polar  staining.  In  old  cultures,  involution  forms 
appear,  as  swollen  coccoid  forms,  or  as  longer  club-shaped 
diphtheroid  forms.  In  the  tissues  they  are  sometimes  found 
to  possess  a  capsule.  They  stain  readily  with  all  the 
usual  aniline  dyes,  dilute  aqueous  fuchsin  and  methylene- 
blue  having  been  mostly  used,  and  these  show  the  polar 
staining  well.  Special  polar  stains  have  been  devised. 
Involution  forms  are  developed  more  rapidly  when  NaCl 
is  added  to  the  medium,  and  "  salt  agar  "  containing  2  to  5 
per  cent  of  NaCl  is  used  for  diagnostic  purposes,  the 
bacilli  showing  the  usual  shape  on  plain  agar,  on  salt 
agar  exhibiting  coccoid,  root-shaped,  large,  globular,  and 
sausage-shaped  forms,  when  the  higher  percentage  is  used  ; 
with  the  lower  percentage,  the  most  striking  feature  is  a 
general  enlargement  of  all  the  bacilli. 

Dr.  R.  M.  Buchanan,  city  bacteriologist  of  Glasgow, 
in  a  Report  to  the  Local  Government  Board  for 
Scotland  ("Thirteenth  Annual  Report  of  the  L.G.B., 
Scotland,  1907,"  page  81),  describes  a  new  culture 
medium  for  B.  pestis  as  follows :  "  Rat  agar  as  a 
culture  medium  for  B.  pestis.  The  susceptibility 
of   the  rat  to  plague  suggested  the  use  of   rat  tissues 


256  PUBLIC    HEALTH    BACTERIOLOGY 

instead  of  ox  flesh  in  the  preparation  of  a  nutrient 
medium  for  the  growth  of  Bacillus  pestis.  An  extract 
is  made  from  the  carcases  of  rats  deprived  of  skin, 
head,  stomach,  and  intestines,  and  the  further  preparation 
of  the  medium  is  essentially  the  same  as  in  that  of  ordinary 
agar,  except  that  the  extract  is  boiled  for  half  an  hour 
before  straining.  Rat  agar  has  been  used  in  the  (city) 
laboratory  for  a  number  of  years  as  a  culture  medium 
for  Bacillus  pestis,  with  most  satisfactory  results,  growth 
taking  place  on  this  medium  with  much  greater  certainty, 
rapidity,  and  profusion  than  on  glycerin  agar.  It  is  also 
noteworthy  that  on  this  medium  the  bacillus  closely 
approximates  to  the  form  which  it  assumes  in  the  body, 
and  is  sometimes  much  elongated.  In  this  elongated 
form  it  may  be  difficult  to  recognize,  so  different  is  it  from 
the  familiar  cocco-bacillary  growth  of  glycerin  agar." 

B.  pestis  is  Gram-negative,  non-gelatin-liquefying,  non- 
motile,  non-sporing,  and  non-indol  forming. 

Cultures. — Grows  readily  and  luxuriantly  on  all  the 
meat-infusion  media.  The  optimum  temperature  is  300  C, 
while  below  200  C.  and  above  380  C.  the  growth  is  sparse 
and  delayed.  The  best  reaction  is  neutrality  or  slight 
alkalinity  ;   but  acidity  does  not  prevent  growth. 

On  agar :  growth  appears  in  twenty-four  hours  as  minute 
colonies,  whitish  and  compact  in  centre,  and  showing  to 
hand  lens  a  broad,  irregular,  indented,  granular  margin. 
Kept  for  a  few  days  at  room  temperature,  some  colonies 
grow  faster  than  others  and  become  more  opaque,  almost 
suggesting  a  mixed  growth  (Muir  and  Ritchie). 

On  gelatin  :  a  similar  growth  occurs  in  two  to  three  days. 
In  stab,  a  white  line  of  growth  takes  place  along  the 
needle  track,  and  little  or  no  surface  growth. 

In  broth :  growth  is  slow,  and  usually  forms  a  slightly 
granular  or  powdery  deposit  at  the  foot  or  sides  of  the  tube 
or  flask.  If  the  surface  is  covered  with  "  ghee  "  (in  India, 
butter  clarified  by  boiling,  and  thus  converted  into  a  kind 
of  oil),  delicate  threads  of  growth  extend  from  the  surface 
downwards,  the  so-called  "  stalactite "  growth,  which 
however  is  not  specific  to  the  plague  bacillus,  nor  is  it 
shown  by  all  races  of  the  organism.  To  observe  it,  the 
culture  must  be  kept  absolutely  at  rest. 


NON-SPORING    BACILLI  257 

Milk  is  not  coagulated.     Litmus  milk  shows  slight  acid 
formation.     Growth   on   potato   and   blood  serum  shows 
nothing  of  differential  value. 
•  In  peptone  water,  no  indol  is  formed. 

Resistance.— Readily  killed  by  heat,  like  all  non-sporing 
forms  ;  ten  minutes  at  650  C.,  or  one  hour  at  580  C.  Drying 
kills  them  in  six  to  eight  days  ;  artificial  drying  in  four 
to  five  hours.  May  live  in  pus  or  sputum  for  eight  to 
fourteen  days.  In  a  moist  dark  place,  may  retain 
viability  for  months  or  even  years.  Freezing  has  little 
effect,  bacilli  surviving  a  temperature  below  o°  C.  for 
forty  days.  Direct  sunlight  kills  them  in  four  to  five  hours  ; 
carbolic  1  per  cent  in  two  hours,  5  per  cent  in  ten  minutes ; 
perchloride  of  mercury  1-1000  in  ten  minutes. 

Pathogenicity. — For  man,  very  pathogenic.  For  animals, 
very  marked  for  rats,  mice,  guinea-pigs,  rabbits,  and 
monkeys.  In  rats  and  guinea-pigs,  the  mere  rubbing  of 
plague  bacilli  into  the  skin  will  often  produce  the  disease, 
and  this  fact  is  made  use  of  in  isolating  the  bacillus  from 
material  contaminated  with  other  bacteria.  Animal 
passage  increases  virulence  ;  growth  on  artificial  media 
diminishes  it,  when  prolonged.  After  subcutaneous  injec- 
tion, a  local  inflammatory  swelling  follows  ;  then  a  swelling 
of  the  corresponding  lymphatic  glands ;  thereafter  a 
general  infection.  Mice  usually  die  in  one  to  three  days, 
rats  and  guinea-pigs  in  two  to  five  days,  and  rabbits  in 
four  to  seven  days.  Post  mortem,  the  chief  changes  are  : 
the  enlarged  glands,  congestion  of  the  organs  (sometimes 
with  haemorrhages),  and  enlargement  of  the  spleen.  The 
bacilli  are  numerous  in  the  lymphatic  glands,  usually  in 
the  spleen,  and  throughout  the  blood.  The  blood  of  a 
plague-stricken  rat  may  contain  as  many  as  100  million 
bacilli  per  c.c. 

Transmission. — Actual  contact  plays  a  very  minor  part 
in  the  transmission  of  the  disease,  as  the  virus  is  not  given 
off  by  the  skin.  The  chief  modes  of  transmission  are  two 
in  number,  by  :  (1)  Inoculation  by  biting  insects  (Limond, 
1899),  tne  usual  insect  being  the  rat  flea  (Pulex  cheopis)  ; 
(2)  Inhalation  :  this  is  the  mode  of  spread  of  the  pneu- 
monic form  of  plague. 

1.  By  inoculation  by  biting  we  get  the  bubonic  plague, 

17 


258         PUBLIC    HEALTH    BACTERIOLOGY 

the  course  of  which  corresponds  exactly  to  that  induced 
in  animals  by  subcutaneous  injection,  as  described  above. 
An  outbreak  of  bubonic  plague  is  always  preceded  by  an 
increased  death-rate  among  rats,  and  that  from  a  disease 
now  known  to  be  due  to  the  B.  pestis.  The  rat  flea  becomes 
infected  by  sucking  the  blood  of  the  rat,  and  infects  other 
rats,  or  it  may  be  human  beings.  It  is  now  believed  that 
the  flea  does  not  inject  the  bacillus  when  biting,  as  no 
bacilli  have  been  found  in  its  biting  apparatus.  It  is 
therefore  surmised  that  the  mode  of  infection  is  by  the 
inoculation  of  the  biting  wound  by  the  rubbing  in  of  the 
excreta  and  vomit  of  the  flea,  both  of  which  are  highly 
charged  with  B.  pestis.  The  proof  that  B.  pestis  multiplies 
in  the  stomach  of  the  flea  is  held  to  follow  from  the  fact 
that  abundant  bacilli  may  be  found  in  it  up  to  twelve  days 
or  longer.  It  has  also  been  observed  that  in  India  plague 
does  not  maintain  itself  in  epidemic  form  after  the  mean 
temperature  has  risen  above  8o°  to  85 °  F.  (26-6°  to  29-4° 
C).  This  has  been  found  to  be  associated  with  a  rapid 
disappearance  of  the  bacilli  from  the  alimentary  canal  of 
infected  fleas  during  the  prevalence  of  this  higher  tempera- 
ture. Transmission  by  inoculation  must  be  extended  to 
the  infection  of  attendants  on  the  sick  and  others,  by 
the  rubbing  in  of  soiled  linen,  etc.,  and  also  from  earth 
soiled  by  the  excreta,  vomit,  and  sputum  of  rats  dead  or 
dying  of  bubonic  or  pneumonic  plague.  Such  transmission 
is  urged  by  some  as  a  likely  one  in  the  case  of  barefoot 
peoples,  living  in  dwellings  with  earth  floors  and  infested 
with  rats.  The  transmission  from  rat  to  rat  is  believed 
to  be  by  the  flea,  in  which  case  the  buboes  are  mainly 
cervical ;  and  by  ingestion  of  rats  dead  of  plague  by 
other  rats,  when  the  buboes  are  mesenteric. 

The  subject  of  transmission  in  this  manner  has  been 
mainly  studied  in  India.  In  Bombay  there  are  two  kinds 
of  rats  :  (1)  Mus  decumanus,  and  (2)  Mus  rattus.  Both 
kinds  are  infested  by  the  same  flea,  the  Pulex  cheopis.  The 
Mus  decumanus  is  the  large  brown  rat,  and  is  the  same 
species  as  that  present  in  Suffolk,  England,  to-day.  The 
Mus  rattus  is  the  black  rat,  and  is  identical  with  the  English 
black  rat.  These  two  species  of  rats  differ  fundamentally 
in  their  habits.     The  Mus  decumanus  is  a  timid  rat,  which 


NON-SPORING    BACILLI  259 

avoids  man  as  far  as  possible,  and  finds  its  food  in  sewers, 
ditches,  fields,  etc.,  rather  than  in  inhabited  houses. 
Mus  rattus,  on  the  other  hand,  is  a  domestic  animal  in 
India,  and  lives  in  close  and  intimate  association  with  the 
home-life  of  the  people.  The  Mus  rattus,  therefore,  is 
chiefly  responsible  for  the  transmission  of  the  disease  to 
man  ;  while  the  Mus  decumanus  is  of  special  importance 
in  maintaining  the  disease  from  season  to  season.  The 
Indian  Plague  Commission  conclude  that  plague  is  a  rat 
disease,  having  a  regular  periodicity,  namely  (a)  an  epizootic 
season,  from  December  to  May  inclusive,  and  (b)t  a  non- 
epizootic  season,  from  June  to  November  inclusive. 
During  the  latter  period  there  are  few  cases  of  plague  in 
rats,  fleas  are  scanty  (this  is  given  as  a  cause  of  the  decrease 
in  cases  of  plague  in  rats),  and  in  some  villages  where  the 
Mus  rattus  alone  prevails,  plague  may  actually  die  out 
each  season.  The  Mus  decumanus  is  more  infested  with 
fleas,  and  is  thought  to  keep  the  infection  going  from 
season  to  season.  A  fresh  epizootic  first  chiefly  affects  the 
Mus  decumanus,  then  spreads  to  Mus  rattus,  and  then  to 
human  beings.  In  this  way  are  explained  the  outbreaks 
of  plague  in  India,  year  after  year  since  1896,  causing 
nearly  a  million  deaths  in  1904  among  the  natives,  while 
the  attendants  and  Europeans  enjoy  almost  complete 
immunity,  although  both  hospitals  and  camps  abound 
with  Pulex  irritans.  This  is  ascribed  to  the  habits  of  this 
common  flea  of  man,  in  that  it  rarely  bites  other  creatures 
than  man,  and  that  in  man  with  plague  the  blood  is  not 
so  alive  with  bacilli  as  in  the  rat.  The  chances  of  infec- 
tion by  Pulex  irritans  are  from  these  causes  enormously 
reduced. 

2.  By  inhalation  of  the  virus,  causing  the  pneumonic 
form  of  the  plague,  which  is  very  rapid  and  fatal.  It  is 
not  understood  what  factors  determine  the  appearance  of 
the  disease  in  this  form,  but  once  started  it  is  extremely 
infectious.  The  symptoms  are  very  similar  to  those  of 
acute  lobar  pneumonia  (although  the  pneumonia  is  mainly 
lobular  in  its  distribution),  with  high  fever,  rapid  respiration, 
and  hemorrhagic  sputa.  The  sputum  contains  the  bacilli 
in  enormous  numbers,  and  almost  in  pure  culture.  In 
Egypt,  the  summer  type  is  bubonic,  and  the  winter  type 
pneumonic. 


260         PUBLIC    HEALTH    BACTERIOLOGY 

Cimex  lectularius,  or  the  common  bed-bug,  has  lately 
been  investigated  as  a  carrier  of  plague  bacilli.  Enormous 
numbers  can  be  found  in  the  stomach  of  the  bug  after 
infection,  and  for  four  to  five  days.  Two  bugs  were  still 
alive  eighty-three  days  after  the  feeding,  and  broth  and 
agar  cultures  were  obtained  from  their  bodies.  Inoculation 
of  mice  produced  typical  results,  and  the  B.  pestis  was 
recovered. 

Other  types  of  plague  are  described,  which  do  not  come 
under  the  above  headings.  These  are  :  the  ambulant  form, 
in  which  the  patient  has  a  few  days  of  fever,  with  swelling 
of  the  glands  of  the  groin,  and  possibly  suppuration. 
These  cases  are  often  found  at  the  beginning  of  an  epidemic, 
and  are  a  source  of  great  danger  to  the  community,  as  the 
urine  and  faeces  contain  bacilli  ;  and  the  septicemic  type,  in 
which  the  patient  succumbs  to  a  virulent  infection  before 
the  buboes  appear.  There  are  also  cutaneous  and  intestinal 
types,  the  former  showing  petechias  and  subcutaneous 
haemorrhages  ;  the  latter,  diarrhoea  and  haemorrhages  from 
the  mucous  membranes,  and  sometimes  the  features  of 
enteric. 

In  India,  an  analysis  of  n,6oo  cases  gave  77-65  per  cent 
of  bubonic  type,  14-25  per  cent  of  the  septicaemic  type, 
and  4-4  per  cent  of  the  pneumonic  type.  The  mortality 
was  highest  in  the  pneumonic  type  (96-69  per  cent),  and 
almost  as  high  in  the  septicaemic.  In  the  bubonic  form, 
out  of  9,500  cases,  5,130  (54  per  cent)  showed  the  glands 
of  the  groin  first  affected,  and  usually  on  the  third  to 
the  fifth  day.*  Resolution  may  occur,  or  suppuration, 
or  in  rare  cases,  gangrene.  Suppuration  is  noted  as  a 
favourable  feature. 

The  appearance  of  petechiae,  or  "  plague  spots,"  or 
"  tokens  of  the  disease,"  gave  to  it  in  the  middle  ages  the 
name  of  the  "  black  death." 

Toxins. — The  filtrate  of  a  plague  culture  has  a  very 
slight  toxic  effect,  but  not  capable  of  inducing  immuniza- 
tion. Hence  it  is  believed  that  little  or  no  soluble  toxin 
exists.     Injection  of  dead  bacilli  produces  distinctly  toxic 

*  The  areas  of  skin  surface  which  drain  respectively  into  the  glands  of  the  groin, 
axilla,  and  neck,  are  as  5  :  1*8  :  1,  and  the  number  of  primary  buboes  observed  in 
hospital  in  these  glands  were  as  5-8  :  1-3  :  1  respectively. 


NON-SPORING    BACILLI  261 

effects.  These  endotoxins  are  comparatively  resistant  to 
heat,  being  unaffected  by  exposure  to  65 °  C.  for  one  hour. 
By  the  injection  of  these  endotoxins  in  suitable  doses,  a 
degree  of  immunity  against  living  virulent  bacilli  is 
obtained.  The  serum  of  such  immunized  animals  is  found 
to  confer  a  degree  of  protection  on  small  animals. 
Immunization. — 

1.  Preventive  inoculation. — Haffkine's  method.  Cultures 
are  made  in  flasks,  with  oil  drops  on  the  surface.  Stalactite 
growths  form,  and  the  flasks  are  shaken  every  few  days 
to  break  those  formed,  and  so  induce  fresh  crops.  The 
incubation  temperature  is  25 °  C,  and  six  weeks'  growth 
is  allowed.  Thereafter  the  culture  is  sterilized  by  heating 
for  one  hour  at  650  C,  and  carbolic  acid  is  added  to  make 
the  bulk  contain  0-5  per  cent.  The  contents  are  well 
shaken  to  distribute  the  sediment,  and  then  bottled  for 
use,  the  fluid  thus  containing  the  dead  bodies  of  bacilli  as 
well  as  any  toxins  that  may  be  in  solution.  It  is 
administered  subcutaneously,  and  usually  in  one  dose  of 
5  c.c.  The  susceptibility  is  said  to  be  reduced  to  one- 
fourth,  and  the  mortality  among  those  inoculated  who 
take  the  disease  is  about  one-half  of  that  among  the 
non-inoculated.  Protection  begins  a  few  days  after  inocu- 
lation and  lasts  for  several  months. 

2.  Anti-plague  Serum. — Yersin  has  prepared  a  serum 
from  horses,  by  injecting  dead  bacilli  into  the  sub- 
cutaneous tissues,  then  into  the  veins,  and  finally,  living 
bacilli  intravenously.  After  a  time,  blood  is  drawn  off,  and 
the  serum  preserved  in  the  usual  way  ;  10  to  20  c.c.  are 
injected  daily.     Some  curative  power  has  been   observed. 

Serum  Diagnosis. — Specific  agglutinins  appear  in  the 
blood  of  some  patients,  but  the  potency  of  the  serum  is 
not  strong,  and  the  test  is  not  easily  carried  out,  owing  to 
the  tendency  of  the  bacilli  to  adhere  in  clumps  preventing 
a  satisfactory  emulsion  being  got.  Hence  the  macroscopic 
or  sedimentation  method  is  preferable.  Cairns,  in  the 
Glasgow  cases,  found  that  the  reaction  appeared  about  a 
week  after  onset  of  the  illness,  increased  until  the  sixth 
week,  and  then  faded  away  ;  being  most  marked  in  severe 
cases  taking  an  early  and  favourable  crisis,  less  in  severe 
cases  tending  to  a  fatal  issue,  and  feeble  or  absent  in  the 
mild  cases.     The  best  dilutions  were  1-10  to  1-50. 


262         PUBLIC    HEALTH    BACTERIOLOGY 

Methods  of  Diagnosis. — 

Bubonic. — (i)  Prepare  the  skin  over  a  bubo,  and  remove 
some  juice  by  aspiration  with  a  sterile  hypodermic  syringe, 
the  needle  of  which  is  plunged  into  the  bubo  ;  (2)  Make 
smears,  and  cultures  on  agar  and  salt  agar  ;  (3)  Inoculate 
a  guinea-pig  with  some  of  the  material,  by  rubbing  it  in  on 
the  shaven  skin  with  a  glass  spatula,  or  by  subcutaneous 
injection. 

In  bubonic  plague,  a  diagnosis  in  many  cases  can  be 
made  by  microscopic  examination  alone,  as  in  no  known 
condition  other  than  plague  do  bacilli  with  the  same 
morphological  characters  occur  in  such  numbers  in  the 
lymphatic  glands. 

Pneumonic. — (1)  Microscopic  examination  of  the 
sputum  ;  (2)  Make  cultures,  inoculate  a  guinea-pig  ;  also 
a  rat  by  smearing  the  sputum  on  its  nasal  mucous- 
membrane. 

In  pneumonic  plague,  a  positive  diagnosis  should  not  be 
given  from  microscopic  examination  alone,  especially  in 
a  plague-free  district,  as  bacilli  morphologically  resembling 
the  plague  organism  may  occur  in  the  sputum  in  con- 
ditions other  than  plague. 

Post  Mortem  of  Rat  Dead  of  Plague. — Subcutaneous 
injection  of  the  flaps  of  the  abdominal  wall  is  noted.  Fluid 
in  the  pleural  cavities,  haemorrhagic  oedema  of  the  neck 
glands,  a  creamy  mottled  appearance  of  the  liver,  and  a 
somewhat  similar  appearance  of  the  spleen.  The  Indian 
investigators  laid  stress  on  an  abundant,  clear,  pleural 
effusion.  The  neck  glands  are  chiefly  involved  in  the 
rat  because  the  flea  prefers  the  skin  of  the  neck  ;  in 
California  and  Glasgow,  however,  the  cervical  bubo  was 
not  so  commonly  found  as  in  India. 

In  chronic  rat  plague,  enlargement  of  the  spleen,  with 
the  formation  of  nodules  in  it  containing  plague  bacilli, 
was  the  usual  finding. 

Prophylaxis. — 

Destruction  of  rats,  by  poisoning,  trapping,  ferreting, 

and  virus. 
Separation  of  rats  from  mankind  by  better  drainage, 

paving   of   yards,    concrete   floors,    and   general 

repairs. 


NON-SPORING    BACILLI  263 

Removal  of  rat  food,  by  frequent  scavenging  and 

attention  to  the  feeding  of  fowls,  pigs,  etc. 
Slaughter-houses  require  special  attention. 
General    campaign    of    cleanliness,     avoidance    of 

fatigue  of  body  or  mind,  and  temperance  in  all 

things. 
Haffkine's  prophylactic  inoculation,  at  least  to  all 

those  on  the  staff  or  otherwise  specially  exposed 

to  infection. 

Nursing. — Bubonic  plague  requires  no  special  pre- 
cautions, as  it  is  not  infectious.  The  liberal  use  of  iodoform 
as  a  dusting  powder,  on  the  person  and  clothing,  is  strongly 
recommended  to  prevent  the  attacks  of  fleas. 

Pneumonic  plague  is  acutely  infectious,  hence  both 
doctor  and  nurse  should  wear  a  cotton-wool  respirator, 
as  should  also  the  attendants. 

Summary. — Plague  is  a  rat  disease.  It  is  conveyed 
from  rat  to  rat,  and  from  rat  to  man,  by  the  rat  flea.  The 
human  flea  is  not  involved  to  any  extent  in  the  matter. 
Bubonic  plague  is  not  an  infectious  disease,  as  this  phrase 
is  commonly  understood.  Pneumonic  plague  is  most 
infectious,  apart  altogether  from  the  question  of  fleas. 
Plague  pneumonias  breed  true,  i.e.,  give  rise  to  other 
cases  of  pneumonic  plague.  The  B.  pestis  blood-count 
is  low  in  man,  high  in  the  rat.  This  affords  an  explana- 
tion of  the  high  degree  of  infectivity  of  the  rat  flea 
as  compared  with  the  human  flea.  Insanitary  conditions 
apart  from  the  presence  of  rats  play  a  secondary  part. 
In  Suffolk,  Mus  rattus  is  rare,  Mus  decumanus  is  common  ; 
therefore  close  contact  with  plague-stricken  rats  is  unlikely, 
and  hence  the  small  epidemic.  (Pringle,  "  The  Outbreak 
of  Rat  Plague  in  Suffolk,"  Public  Health,  January,  1911.) 

For  an  important  article  on  the  "  Spread  of  .Plague,"  by 
C.  J.  Martin,  and  the  subsequent  discussion,  see  Brit.  Med. 
Jour.,  1911,  vol.  ii,  p.  1249. 

Summary  of  the  "  Lancet  "  Reports  on  the  Plague  in 
China,  1910-1911. 

The  outbreak  of  plague  in  China,  beginning  in  Manchuria 
on  October  12th,  1910,  and  extending  rapidly  until  it  had 


264         PUBLIC    HEALTH    BACTERIOLOGY 

invaded  widely  separated  districts,  was  notable  in  several 
important  particulars.  The  epidemic  was  almost  without 
exception  one  of  primary  pneumonic  plague.  The  fatality 
was  extremely  high,  few  cases  of  recovery  having  been 
reported.  The  infectious  nature  of  the  malady  was  very 
great,  and  the  virus  was  apparently  carried  by  the  sputum. 
The  origin  of  the  plague  was  not  ascribed  to  rats,  but  to 
marmots,  a  species  of  squirrel  living  in  burrows.  The 
question  of  infection  by  fleas  is  here  of  minor  importance, 
once  the  epidemic  is  started.  Its  relation  to  the  origin  of 
the  epidemic  has  not  been  worked  out.  Once  begun,  the 
propagation  was  apparently  by  direct  inhalation  of  the 
virus. 

The  Chinese  Government  invited  the  other  Governments 
to  send  representatives  to  an  International  Plague 
Conference,  which  began  its  sittings  at  Mukden  on  April 
3rd,  191 1.  The  following  statements  are  taken  from  the 
reports  of  the  proceedings  published  in  the  Lancet  from 
April  29th,  191 1,  onwards,  which  include  an  exhaustive 
report  by  Dr.  G.  Douglas  Gray,  physician  to  H.B.M. 
Legation,  Peking,  the  questions  for  discussion,  and  the 
Chairman's  inaugural  address. 

Origin. — It  had  been  known  for  many  years  that  in 
Eastern  Siberia  and  Mongolia  the  marmot,  or  tarabagan 
(Russian),  or  han  ta  (Chinese),  a  variety  of  the  squirrel 
tribe,  of  the  rodent  genus,  frequently  surfers  from  a  fatal 
disease  which  may  be  transmitted  to  man  and  produce 
symptoms  indistinguishable  from  bubonic  and  pneumonic 
plague.  This  animal  is  hunted  for  its  fur,  which  is  used 
to  imitate  sable  and  other  furs.  One  of  its  favourite 
haunts  is  a  mountain  range  in  the  north-west  of  Man- 
churia, and  here  large  numbers  of  Chinese  are  employed 
trapping  it  during  the  summer  months.  It  was  among 
these  trappe*rs  that  the  present  outbreak  is  held  to  have 
originated.  The  proof  that  the  "  tarabagan  disease  "  in 
man,  and  plague,  were  one  and  the  same  disease,  has 
not  been  given  bacteriologically,  and  in  1905,  1906,  and 
1907,  Russian  scientific  expeditions  were  sent  to  investi- 
gate and  report.  Dr.  M.  T.  Schreiber  concluded  that  : 
(1)  Epizootics  (a  term  applied  to  those  animal  diseases 
which   behave    as   epidemics  do    in  the    human    species) 


NON-SPORING    BACILLI  265 

undoubtedly  do  occur  without  human  beings  becoming 
infected  ;  (2)  Field  mice  do  not  contract  the  disease  from 
the  tarabagan,  though  they  have  every  chance  of  doing  so, 
and  are  known  to  be  susceptible  to  plague  ;  (3)  Domestic 
animals  also  escape  it,  although  dogs  eat  the  flesh  of  the 
dead  marmot. 

Dr.  B.  A.  Barykin  reported  in  1909  the  following  facts  : 
In  1906  the  marmots  around  a  settlement  called  Abogaitui 
showed  a  high  mortality  from  spring  to  autumn,  but  the 
inhabitants  were  aware  of  the  danger  and  avoided  all 
contact  with  the  sick  animals,  except  a  Cossack,  who 
was  in  indifferent  health  and  had  a  craving  for  the 
flesh  of  a  tarabagan.  He  got  some,  fell  ill  with  the 
symptoms  of  plague,  and  died  in  four  days.  Others 
became  ill,  and  in  all  eight  died  with  symptoms  of 
pneumonic  plague.  Post  mortems  were  held  in  two  cases, 
and  bacilli  indistinguishable  from  the  plague  bacilli  were 
found  in  the  organs  ;  and  mice  injected  with  the 
splenic  juice  died  in  twenty-six  hours  with  the  typical 
appearances.  This  was  in  September,  1906.  In  the 
autumn  of  1907,  marmots  were  caught  or  killed  by  the 
party  and  examined  for  the  presence  of  disease.  In  one 
of  these  animals  showing  no  external  signs  of  being  ill 
save  some  degree  of  malnutrition,  the  spleen  was  found 
to  be  swollen  -and  congested,  and  contained  large  numbers 
of  bacilli  identical  in  morphology  and  culture  with  the 
plague  bacilli.  A  fortnight  later,  twelve  miles  from  where 
this  animal  was  discovered,  the  men  of  an  isolated  Cossack 
family,  hunting  the  marmots  around  them,  killed  one 
showing  clear  signs  of  illness.  In  spite  of  the  counsel  of 
the  elder  men  the  animal  was  skinned  and  the  body  was 
given  to  a  girl  of  thirteen  years  to  take  to  the  fields.  She 
dragged  its  body  (said  to  be  15  lb.  weight)  after  her 
through  the  grass,  and  returned  barefoot  over  the  same 
path.  On  the  next  day  she  fell  ill,  a  bubo  appeared  in 
the  left  groin,  and  she  died  some  days  later  with  all  the 
symptoms  of  plague.  From  the  bubo,  from  a  pustule  on 
a  finger,  and  from  the  spleen,  bacilli  indistinguishable  from 
plague  bacilli  were  isolated,  and  being  injected  into  mice 
caused  their  death  in  eighteen  hours,  of  septicaemia. 
Neither  the  body  nor  the  skin  of  the  animal  was  recovered. 


266         PUBLIC    HEALTH    BACTERIOLOGY 

The  rest  of  the  family  escaped.  The  same  autumn  a  railway 
guard  who  had  caught  marmots,  and  a  woman  who  had 
skinned  them,  both  died  of  a  disease  resembling  bubonic 
plague.  In  the  guard,  typical  bacilli  were  found.  A 
history  of  ten  outbreaks  in  nine  years  in  the  district  around 
one  railway  station  was  elicited.  Various  other  localized 
outbreaks  were  reported,  all  tending  to  show  that  plague 
was  not  new  to  the  districts  where  Mongolia,  Manchuria, 
and  Siberia  adjoin. 

Spread. — Beginning  then  in  the  north-west  borders  of 
Manchuria  among  the  marmot  hunters,  it  was  carried  by 
these  in  travelling  back  to  their  homes,  many  of  them 
having  come  from  the  Shantung  province,  south  of  Peking. 
In  the  third  week  of  October,  1910,  about  10,000  of  these 
men  had  gathered  in  Manchourie  and  Khailar,  stations  on 
the  Vladivostock  railway,  waiting  to  sell  the  skins  they 
had  gathered  and  to  return  to  the  south  for  the  winter  and 
the  new  year  festival.  Cases  of  illness  occurred  here,  with 
symptoms  of  headache,  fever,  spitting  of  blood-coloured 
sputum,  and  followed  by  rapid  death.  Apparently  in  spite 
of  the  risks  not  many  hunters  die  on  the  plains  ;  but 
when  crowded  into  the  poor  hovels  or  inns  of  the  market- 
towns, — where,  in  small  badly  ventilated  rooms,  twenty  to 
forty  may  be  found  living,  sleeping,  and  eating  beside  piles 
of  raw  pelts — the  conditions  for  the  encouragement  of 
any  epidemic  disease  are  ideal.  From  these  foci  the 
infection  spread  by  railway  trains  in  which  the  hunters 
were  carried  to  Harbin,  then  south  to  Chang-chun,  thence 
to  Mukden,  on  to  Tientsin,  and  from  there  to  their  home 
villages  in  the  Shantung  province ;  and  also  by  foot 
travellers  who  struck  across  country  through  Kirin  city 
{eighty  miles  from  a  railway)  to  Dalny,  and  thence  by 
boat  to  Chef 00,  a  port  in  the  Shantung  province.  All 
the  way  men  were  falling  ill  and  dying,  and  the  sick 
and  dead  were  thus  deposited  at  all  the  places  passed 
en  route.  The  infection  reached  Harbin  on  November 
7th,  and  in  three  months  there  were  5,000  deaths  out 
of  a  population  of  30,000.  Two  factors  seem  to  have 
contributed  largely  to  the  virulence  of  the  epidemic  in 
the  Chinese  city.  First,  the  severe  climatic  conditions,  the 
thermometer  registering  at  times  —  300  C.  (  —  22°  F.,  or  54 


NON-SPORING    BACILLI  267 

degrees  of  frost),  which  prevented  the  people  going  out  of 
doors.  Secondly,  the  low,  dark,  dirty,  and  overcrowded 
houses  which  form  the  majority  of  the  dwellings.  Never- 
theless in  Shuangcheng  Fu,  a  finely  planned  city  with  wide 
streets,  spacious  compounds,  and  well-constructed  houses, 
and  with  but  little  poverty,  there  were  1,500  deaths  in 
seven  weeks  out  of  60,000  inhabitants. 

Symptoms. — The  incubation  period  in  the  majority  of 
cases  was  five  days.  There  were  no  marked  prodromal 
symptoms.  Often  a  man  had  a  normal  pulse  and 
temperature  on  one  day  and  was  dead  the  next.  The 
invasion  was  without  rigor,  with  feelings  of  illness,  weak- 
ness, and  giddiness.  A  sudden  onset  with  headache,  then 
bloated  face  and  suffused  conjunctivae  (septicaemic  cyanosis), 
with  temperature  over  1030  F.,  and  fast  fluttering  pulse, 
was  usual.  The  respirations  averaged  thirty-five  per 
minute.  Coarse  crepitant  rales  were  noted  all  over  the 
chest,  but  little  or  no  impairment  of  resonance.  These 
rales  are  due  to  marked  oedema  of  the  lungs  in  the  late 
stages  of  the  disease.  In  the  earlier  stages  rales  are  rarely 
present  even  in  serious  cases,  and  then  they  are  usually 
fine.  Blood-stained  sputum  is  often  the  first  sign  of 
illness  in  pneumonic  cases.  The  signs  of  cardiac  involve- 
ment are  always  marked  in  advanced  cases :  very  rapid 
feeble  running  pulse,  agonizing  dyspnoea,  galloping  rhythm 
of  the  heart  sounds,  and  sudden  heart  failure.  Death 
occurs  from  the  intoxication,  with  paralysis  of  the  heart. 
Death  resulted  in  attempts  to  move  patients  and  where 
the  patients  sat  up  in  bed  for  a  few  minutes  to  take  nourish- 
ment. Labial  herpes  was  not  observed  in  any  of  the 
patients  seen  in  hospital,  which  is  a  point  noted  before 
and  interesting  in  comparison  with  acute  lobar  pneumonia, 
in  which  it  frequently  occurs.  In  the  septicaemic  form 
there  may  be  a  flow  of  blood  from  the  nose  or  mouth 
shortly  before  death.  No  glandular  enlargements  were 
noted  except  once  at  Harbin,  where  there  was  a  sub- 
maxillary bubo  followed  by  secondary  plague  pneumonia 
and  death.  The  bacteriological  diagnosis  was  the  only 
certain  one,  as  the  symptoms  were  so  variable.  Many 
were  able  to  walk  about  until  within  a  few  hours  of  death, 
and  up  to  that  time  declaring  that  they  were  quite  well. 


268         PUBLIC    HEALTH    BACTERIOLOGY 

Etiology  and  Pathology. — Dr.  R.  P.  Strong  and  Dr.  Teague 
performed  twenty-five  post-mortem  examinations,  and 
came  to  the  following  conclusions  :  That  epidemic  plague 
pneumonia  results  from  inhalation,  the  primary  point  of 
infection  being  the  bronchi.  Through  the  bronchi  the 
bacilli  reach  the  lung  tissue,  and  rapidly  multiplying  there 
they  produce  pneumonic  changes  of  the  lobular  type  and 
later  more  general  lobar  involvement.  The  blood  becomes 
quickly  infected  and  a  true  bacteriaemia  results  in  every 
case.  Secondary  pathological  changes  occur,  especially  in 
the  spleen,  bronchial  glands,  heart,  blood-vessels,  kidneys, 
and  liver.  The  fact  that  the  bronchial  glands  at  the 
bifurcation  of  the  trachea  are  always  much  more  affected 
than  any  of  the  other  lymphatics,  argues  against  the  theory 
that  epidemic  plague  pneumonia  is  primarily  a  septicaemia 
with  secondary  involvement  of  the  lungs.  Moreover,  in 
the  earliest  stages  of  the  disease,  the  blood  may  be  free  of 
plague  bacilli.  The  condition  observed  in  the  trachea  and 
bronchi  in  epidemic  plague  pneumonia  is  pathognomic  of 
this  condition  alone.  The  throat  and  larynx  may  show 
characteristic  appearances  at  times.  The  tonsils  may 
become  secondarily  infected  like  other  lymph  follicles,  but 
the  duration  of  the  disease  is  too  short  to  allow  of  this  as  a 
rule.  Primary  infection  by  tonsil  can  occur,  with  enlarge- 
ment of  the  glands  of  the  neck  early  in  the  disease.  The 
oesophagus  was  found  normal  in  every  case,  which  argues 
against  primary  intestinal  plague  infection,  since  plague 
bacilli  must  have  been  repeatedly  swallowed  in  sputum 
and  bronchial  secretion  in  many  of  the  cases. 

Dr.  Koulecha,  who  had  dissected  twenty-eight  plague 
corpses,  read  a  communication  in  which  he  differed  in 
toto  from  the  above  in  regard  to  the  mode  of  infection. 
From  the  necropsies  and  microscopical  examination  of  the 
tissues  he  concludes  that  pneumonic  plague  is  a  septicaemic 
disease,  in  which  an  overflooding  of  the  blood  and  the 
lymphatic  system  with  bacilli  could  be  observed. 
Infectious  matter  entered  the  mouth,  affecting  en  route 
the  tonsils,  mucous  membrane  of  the  trachea,  bronchi, 
and  neighbouring  lymphatic  glands  ;  and  from  these  the 
bacilli  passed  into  the  blood.  The  lungs  were  apparently 
affected  secondarily  from  the  blood,  which  he  inferred  from 


NON-SPORING    BACILLI  269 

the  great  accumulation  of  the  plague  bacilli  in  the  peri- 
vascular spaces.  On  this  view,  pneumonic  plague  is  a 
lobar  pleuro-pneumonia  of  hematogenous  origin,  and 
should  be  classed  with  croupous  pneumonia.  Dr.  Fujinami 
confirmed  these  findings.  Professor  Zabolotny  observed 
that  it  had  not  been  sufficiently  proved  how  many  were  of 
direct  pulmonary  origin  and  how  many  were  of  hemato- 
genous origin. 

Rats  and  Fleas. — Professor  Kitasato  reported  that  of 
30,000  rats  examined  in  South  Manchuria,  6  per  cent  were 
Mus  rattus,  but  none  of  the  30,000  showed  plague  infection. 
In  North  China,  of  3,000  rats  examined  living  by  Dr 
Andrew,  all  were  Mus  decumanus  ;  he  had  never  found 
Mus  rattus.  He  had  noted  a  seasonal  flea  prevalence, 
highest  in  September,  October,  and  November.  The  only 
species  noted  was  Pulex  cheopis.  Dr.  Petrie  found  both 
Pulex  cheopis  and  Ceratophyllus  unisus  among  fleas 
examined  at  Mukden.  Dr.  Petrie  also  examined  twelve 
tarabagans  sent  direct  from  Manchuria  to  Mukden.  On 
these  he  found  thirty-five  fleas,  twelve  being  on  one 
alone.  (Ticks  were  also  observed  on  them.)  The  fleas 
found  were  unusually  large,  and  on  superficial  examination 
appeared  to  belong  to  the  genus  Histricopsylla  (eyeless, 
truncated  head,  comb  on  the  inferior  border  of  head, 
thorax,  and  part  of  abdomen,  numerous  long  hairs  over 
the  body). 

In  an  epidemic  of  plague  in  Tongshan,  in  1908,  bubonic 
cases  predominated  at  the  first,  but  the  proportion  of 
septicemic  and  pneumonic  gradually  increased.  Of  the 
rats  examined,  1  per  cent  were  found  to  be  affected. 
Some  rats  which  remained  well  for  days  without  any  sign 
of  glandular  enlargement  during  life,  were  found  post 
mortem  to  have  Bacillus  pestis  in  the  splenic  blood,  and 
were  evidently  immune  to  infection,  at  least  for  the  time 
being.  Rats  trapped  in  the  mines  in  the  neighbourhood 
were  found  to  be  less  readily  infected  experimentally 
than  those  found  above  ground.  The  death-rate  in  the 
Tongshan  epidemic  was  800  out  of  1000  cases. 

Diagnosis. — Sputum  is  scanty  at  first,  and  the  bacilli 
are  difficult  to  find  in  it.  Later,  it  is  almost  a  pure  culture. 
Professor  Zabolotny  advised  Gram's  method  for  staining 


270         PUBLIC    HEALTH    BACTERIOLOGY 

the  sputum.  He  had  often  noted  mixed  infections,  and  a 
Gram-negative  bi-polar-staining  bacillus  was  seen  at  times, 
which  was  not  the  plague  bacillus.  It  required  further 
study.  Involution  forms  bore  no  relation  to  virulence. 
Blood  cultures  could  usually  be  got  forty-eight  hours 
before  death.  At  least  i  c.c.  of  blood  must  be  used  for 
the  test.  In  this  way  an  earlier  diagnosis  could  be  made 
than  by  the  sputum.  In  recovered  bubonic  cases,  agglu- 
tination could  be  got  in  the  second  and  third  weeks  with 
dilutions  of  1-25  and  1-50.  Agglutination  and  fixation 
of  the  complement  experiments  were  of  little  value  in 
pneumonic  plague.  Dr.  Broquet  advised  the  use  of  the 
following  solution  for  the  conservation  of  suspicious 
plague  organs  for  future  study  or  transmission  to  a 
laboratory  at  a  distance :  neutral  glycerin,  20  c.c, 
calcium  carbonate,  2  grm.,  distilled  water,  80  c.c.  Mix  at 
300  C.,  and  immerse  tissues. 

Vaccination. — Protective  vaccination  with  attenuated 
plague  bacilli  was  advocated  by  Dr.  Strong.  Professor 
Galeotti  advised  the  vaccine  of  Lustig  and  Galeotti  for 
these  reasons  :  (1)  The  toxin  (Galeotti  holds  that  it  is  an 
endotoxin  and  non-soluble  ;  Zabolotny  holds  that  it  is  a 
soluble  toxin),  and  no  other  substance,  is  used  ;  (2)  It  can 
be  dried  and  standardized  ;  (3)  The  plague  nucleo-proteid 
can  be  stored  in  a  sterile  condition.  The  general  experience 
was  that  no  form  of  vaccination  gave  much  protection 
against  pneumonic  plague. 

Serum  Therapy. — Dr.  Martini  advised  passive  immuniza- 
tion for  all  those  exposed  to  infection,  such  as  doctors  and 
nurses,  etc.  Small  doses  were  useless  ;  100  c.c.  at  least 
must  be  used,  and  repeated  soon.  As  regarded  the  present 
epidemic,  he  thought  the  protective  value  was  small. 
Professor  Zabolotny  reported  that  he  had  used  up  to 
1  litre  of  serum  without  success,  only  prolonging  the  illness. 
Dr.  Paul  Haffkine  had  seen  protective  effect  after  large 
doses,  but  this  did  not  last  more  than  five  days. 

Quarantine. — The  use  of  railway  cars  for  this  purpose 
was  a  new  feature.  This  gives  a  segregation  camp  divided 
into  small  units,  each  completely  isolated  from  the  other, 
easy  of  disinfection,  easy  to  supervise  and  for  the  detection 
of    onset    of  sickness.     There  were   3000    suspects  thus 


NON-SPORING    BACILLI  271 

isolated  at  Harbin,  the  railway  cars  being  drawn  into 
specially  constructed  sidings. 

Disposal  of  the  Dead. — With  the  ground  frozen  at  —  40 °  C, 
ordinary  burial  of  such  large  numbers  of  bodies  was  not 
easy.  In  spite  of  Chinese  traditions,  the  Government 
gave  orders  for  the  cremation  of  the  bodies  of  those  dead 
of  plague.  This  was  carried  out  thus  at  Harbin :  A  pit 
20  feet  square  by  10  feet  deep  was  made  by  blasting  with 
dynamite.  When  bodies  were  in  coffins,  these  sufficed  to 
aid  the  burning  ;  but  if  they  were  not,  four  pieces  of 
round  wood  4  inches  in  diameter  by  2  feet  long,  were 
added  per  body.  The  pit  held  about  500  bodies,  and 
into  it  kerosene  was  pumped  from  a  fire-engine  ;  about 
10  gallons  per  100  bodies.  On  being  set  alight  the 
mass  burned  fiercely  and  rapidly,  and  little  but  ashes 
remained. 

End  of  Epidemic. — The  epidemic  seemed  to  die  out 
(April,  1911)  when  the  temperature  rose  to  —  200  C. 
(  —  40  F.).     The  total  mortality  was  over  40,000. 

Summary  of  Conclusions. — The  disease  spread  by  direct 
infection  from  man  to  man.  Whatever  may  have  been 
its  primary  origin,  there  is  no  evidence  that  a  concurrent 
epizootic  in  rodents  played  any  part  in  its  wide  dissemina- 
tion. From  Russian  sources  reports  of  an  epizootic 
disease  among  tarabagans  have  been  received,  and  it  is 
not  unlikely  that  this  is  plague,  but  the  bacteriological 
proof  is  not  yet  complete.  The  infection  was  introduced 
into  towns  and  villages  by  persons  actually  suffering  from, 
or  by  those  in  the  incubation  stage  of,  the  disease.  There 
has  been  no  positive  epidemiological  evidence  to  show 
that  the  disease  has  been  spread  by  clothing,  merchandise, 
or  other  inanimate  objects.  So  far  as  can  be  ascertained 
the  only  infective  agent  in  the  epidemic  has  been  the 
sputum  of  the  plague  patient.  In  the  majority  of  the 
cases  the  disease  has  been  contracted  by  the  inhalation 
of  plague  bacilli  in  droplets  of  sputum,  causing  infection 
of  the  lower  portion  of  the  trachea  and  of  the  bronchi. 
In  infection  by  inhalation  the  risk  to  the  person  exposed 
bears  a  direct  relation  to  his  proximity  to  the  patient 
and  to  the  duration  of  the  exposure.  In  view  of  this, 
masks  and  goggles  should  be  worn  by  all  who  come  in 


272         PUBLIC    HEALTH    BACTERIOLOGY 

contact  with  cases  of  the  disease  or  suspected  cases.  The 
best  form  of  mask  is  a  three-tailed  gauze  bandage  with 
a  pad  of  cotton-wool.  It  should  be  destroyed  or  dis- 
infected after  each  exposure  to  infection.  The  epidemic 
was,  almost  without  exception,  one  of  primary  pneumonic 
plague,  with  an  incubation  period  of  from  two  to  five  days. 
An  increased  temperature  and  pulse-rate  are  usually  the 
earliest  signs  observable,  but  a  diagnosis  cannot  be  made 
until  the  organisms  are  recognized  in  the  characteristic 
blood-stained  sputum. 

An  accurate  diagnosis  can  only  be  made  by  a  bacterio- 
logical examination  of  the  sputum  to  exclude  pneumonic 
infection  due  to  other  micro-organisms.  Since  the  evidence 
points  to  the  conclusion  that  in  the  epidemic  all  the  cases 
became  septicemic,  an  examination  of  the  blood  micro- 
scopically or  culturally  may  be  a  valuable  aid  in  diagnosis. 
The  physical  signs  of  lung  involvement  are  too  indefinite 
and  appear  too  late  in  the  course  of  the  disease  to  be  of 
diagnostic  value,  and  even  in  cases  in  which  the  condition 
of  the  patient  is  grave  they  may  be  very  slight. 

The  fatality  has  been^  extremely  high,  scarcely  any 
recovering.  The  general  experience  has  been  that  no 
method  of  treatment  has  been  of  avail  in  saving  life,  but 
that  the  serum  treatment  seems  in  a  few  instances  to  have 
prolonged  it.  The  decline  of  the  epidemic  has  not  been 
due  to  any  loss  of  virulence  of  the  bacillus,  but  probably 
to  the  preventive  measures  which  were  enforced  either  in 
accordance  with  scientific  methods  or  by  the  efforts  of 
the  people  to  protect  themselves. 

The  statistics  of  prophylactic  inoculation  collected 
during  the  past  epidemic  do  not  allow  of  any  definite 
conclusion  being  formed  about  their  value  in  plague 
pneumonia  ;  but  in  bubonic  plague  it  was  argued  that 
some  degree  of  protection  is  conferred  by  the  use  of 
vaccines.  Further  experiments  in  animals  are  recom- 
mended, in  reference  to  securing  immunity  against 
pneumonic  plague  infection.  {Lancet,  191 1,  vol.  I, 
pp.  1614-16.) 


NON-SPORING    BACILLI  273 


THE     TUBERCLE     BACILLUS, 

This  bacillus  is  the  cause  of  tuberculosis,  an  infective 
disease,  characterized  by  lesions  which  are  nodular  bodies 
called  "  tubercles,"  or  by  diffuse  infiltrations  of  tuberculous 
tissue,  which  may  undergo  caseation  and  finally  ulcerate, 
or  may  become  sclerosed,  and  in  some  cases  calcified. 
The  infectious  nature  of  tuberculous  material  was  for  long 
suspected,  was  proved  by  Villemin  in  1865,  and  by  Armanni 
and  Cohnheim  and  Salomonsen  from  1870  to  1880. 
Baumgarten  first  described  the  bacillus  in  sections,  but 
Koch  first  established  its  causal  nature  on  a  solid  basis 
by :  (1)  Demonstrating  the  presence  of  the  tubercle 
bacillus  in  a  great  variety  of  tissues  and  organs  ;  (2)  Pre- 
paring pure  cultures  of  the  organism  from  these ; 
(3)  Producing  the  disease  by  the  inoculation  of  the  bacillus 
derived  from  pure  culture  ;  and  (4)  Recovering  the  same 
organism  from  the  diseased  parts  of  inoculated  animals. 
These  four  articles  are  known  as  Koch's  postulates,  and 
all  of  them  have  to  be  satisfied  before  an  organism 
can  be  absolutely  proved  as  the  cause  of  a  specific 
disease.  At  present,  for  quite  a  number  of  bacteria, 
some  of  these  postulates  have  yet  to  be  fulfilled  as 
regards  mankind. 

Description. — Bacillus  tuberculosis  is  a  slender  rod,  often 
slightly  curved,  measuring  2-5  to  3-5  micra  long  by  0-3 
micron  in  width.  They  are  of  uniform  thickness,  or  may 
show  slight  swelling  at  the  ends  or  even  in  their  length. 
They  may  lie  singly  in  the  tissues  or  sputum,  but  often  in 
small  heaps  or  masses.  In  cultures,  a  remarkable  fila- 
mentous growth  has  been  repeatedly  observed.  In  the 
sputum,  long  branching  hypha-like  filaments,  sometimes 
with  swollen  ends,  have  been  found.  These  are  looked 
upon  by  some  bacteriologists  as  involution  forms,  but  many 
regard  them  as  evidence  of  the  relationship  of  the  tubercle 
bacillus  to  the  hyphomycetes.  A  capsular  or  enveloping 
substance  is  produced  by  the  bacillus,  more  by  the  human 
form  than  by  the  bovine,  and  more  the  longer  the  growth 
on  blood  serum.  When  stained,  the  bacillus  often  appears 
beaded,  the  unstained  spaces  being  regarded  as  vacuoles 
by  some  and  as  spores  by  others.     Inasmuch  as  the  bacilli 

18 


274         PUBLIC    HEALTH    BACTERIOLOGY 

showing  these  have  no  increased  resistance  against  heat 
and  disinfectants,  the  spore  interpretation  is  probably 
incorrect.  The  beads  or  highly  stained  portions  have 
likewise  been  called  spores,  but  the  whole  matter  is  at 
present  unsettled. 

The  tubercle  bacillus  stains  imperfectly  or  not  at  all 
with  ordinary  watery  aniline  dyes,  and  only  after  long 
exposure  or  heating,  or  more  quickly  if  a  mordant  is  used. 
Once  stained,  the  bacilli  retain  the  dye  tenaciously,  in 
spite  of  treatment  with  alcohol  or  strong  acids,  and  for  the 
latter  reason  they  are  spoken  of  as  "  acid-fast  "  bacilli  (acid- 
proof  would  be  a  better  term).  This  feature  seems  to  be 
due  to  the  presence  of  fatty  substances  in  the  cell,  and 
has  furnished  the  basis  for  differential  staining  methods. 
The  fatty  substances  are  really  wax-like  in  nature,  and  are 
soluble  in  alcohol  +  ether. 

B.  tuberculosis  is  non-motile,  non-nagellar,  non-sporing, 
does  not  grow  on  gelatin,  and  is  Gram -positive.  Recently 
varieties  have  been  described  which  are  not  acid-fast,  but 
are  stained  by  Gram's  method  prolonged,  and  these  forms 
are  stated  to  be  present  in  old  tuberculous  lesions,  where 
the  ordinary  form  is  not  found  and  yet  the  material  is 
virulent.  These  are,  (i)  A  fine  bacillary  form,  often 
showing  granules,  and  (2)  Free  granules.  Much  also 
found  that  when  acid-fast  forms  were  added  to  milk 
(sterilized)  and  incubated,  the  acid-fast  forms  disappeared, 
and  yet  when  the  milk  was  injected  into  an  animal,  tuber- 
culosis was  produced  and  in  the  lesions  acid-fast  bacilli 
were  demonstrable.  If  these  statements  are  conclusively 
proved,  they  are  of  the  first  importance. 

Chemical  Analysis  of  Tubercle  Bacilli. — Water  85-9  per 
cent,  solids  14-1  per  cent.  The  ash  shows  55  per  cent  of 
P205,  12-6  per  cent  of  CaO,  11-5  per  cent  of  Mg,  13-6  per 
cent  of  Na20,  6.3  per  cent  of  K20. 

Cultures. — The  bacillus  is  not  easily  cultivated  ;  growth 
fails,  or  is  slow  or  scanty  ;  fails  on  agar  and  gelatin  entirely, 
but  on  glycerin  agar  (3  to  6  per  cent)  and  in  glycerin  broth 
(6  per  cent)  growth  takes  place  in  ten  to  fifteen  days, 
becoming  visible  first  as  dry  white  spots,  the  extension 
and  fusion  of  which  form  a  dull,  whitish,  wrinkled  pellicle 
or  layer.     These  media  are  suitable  for  subcultures ;    for 


NON-SPORING    BACILLI  275 

growth  direct  from  the  tissues,  blood  serum  and  Dorset's 
egg  media  are  commonly  used. 

On  blood  serum,  at  37-5°  C,  growth  appears  in  ten  to 
fourteen  days  as  minute  spots,  rather  irregular,  raised 
above  the  surface,  and  comparable  to  small  dry  scales. 
The  culture  is  got  by  inoculating  with  a  sterile  platinum 
loop  from  foci  in  a  tuberculous  gland ;  or  by  planting  an 
actual  piece  of  tissue  on  to  the  surface  of  the  serum ;  or 
by  inoculating  with  tubercle  bacilli,  derived  by  the 
anti-formin  method  (see  later)  from  sputum  or  digested 
tissue. 

On  Dorset's  egg  media,  vigorous  growth  takes  place 
resembling  that  on  blood  serum.  To  make  the  medium : 
take  the  whole  contents  of  four  eggs,  beat  well,  add  25  c.c. 
of  water,  and  mix  thoroughly ;  filter  through  muslin  to 
remove  air  bells ;  fill  into  tubes,  and  heat  these  in 
sloped  position  for  four  hours  at  700  C.  They  are 
now  ready  ;  but  before  inoculation,  two  drops  of  steri- 
lized water  are  placed  on  the  surface  of  the  medium. 
When  inoculating,  the  material  is  well  rubbed  over  the 
surface,  the  plug  is  replaced,  and  is  sealed  over  with  a 
few  drops  of  paraffin,  and  the  tube  incubated  in  the 
sloped  position. 

On  glycerin  potato,  growth  takes  place,  and  on  other 
purely  vegetable  media. 

Optimum  Conditions. — B.  tuberculosis  is  markedly  aerobic. 
It  grows  at  human  blood-heat,  370  to  380  C.  The  usual 
range  is  280  to  420  C.  but  it  can  be  acclimatized  to  grow 
at  220  to  23 °  C.  In  fluid  media  it  is  killed  in  fifteen  to  twenty 
minutes  at  6o°  C,  in  five  minutes  at  8o°  C,  and  in  one  to 
two  minutes  at  900  C.  It  can  resist  dry  heat  at  ioo°  C.  for 
one  hour.  Simple  drying  is  not  efficient,  as  still  virulent 
forms  have  been  found  in  dried  tuberculous  sputum  after 
two  months.  Similarly,  putrefaction  of  sputa  or  tissue  does 
not  destroy  the  bacilli  readily,  for  they  have  been  found 
aliyo  under  such  conditions  after  three  weeks.  Gastric 
juice  failed  to  kill  them  in  six  hours,  and  several  three- 
hour  spells  of  freezing  at  —  30  C.  had  little  effect.  Direct 
sunlight  rapidly  kills  them  ;  5  per  cent  carbolic  kills  them 
in  a  few  minutes,  but  if,  as  in  sputum,  they  are  protected 
by  mucus,  complete  disinfection  takes  five  to  six  hours. 


276         PUBLIC     HEALTH    BACTERIOLOGY 

Perchloride  of  mercury  is  not  very  efficient,  from  the 
albuminate  formed.  The  comparatively  high  powers  of 
resistance  (for  an  organism  not  admitted  to  have  spores) 
are  attributed  to  the  protective  qualities  of  the  waxy  cell 
membrane.  The  conclusion  then  is,  that  moist  heat  at 
ioo°  C.  will  kill  the  bacilli  in  fluids  and  tissues,  provided 
time  is  given  for  penetration  of  bulky  materials.  In 
Germany,  tuberculous  ox  flesh  is  thus  sterilized  (four  hours 
boiling)  and  then  allowed  to  be  sold. 

Pathogenicity. — For  man  is  great :  10  per  cent  of  all  the 
deaths  in  Great  Britain  and  in  the  United  States  of  America 
are  due  to  tuberculosis  of  one  kind  or  another.  This  of 
course  represents  only  a  portion  of  the  incidence  of  tuber- 
culosis, as  many  people  are  attacked  and  succeed  in  throw- 
ing off  the  infection.  In  fact  it  may  be  said  that  the  fatal 
attack  of  tuberculosis  is  in  many  cases  only  the  last  of  a 
long  series  of  attacks  more  or  less  successfully  repulsed. 
The  commonest  type  in  man  is  phthisis  pulmonale  s,  then 
affections  of  lymphatic  glands,  bones  and  joints,  and  serous 
membranes. 

In  America,  22  per  cent  of  the  deaths  among  the  North 
American  Indians,  and  16  per  cent  among  the  negroes, 
are  due  to  tuberculosis. 

For  animals,  the  pathogenicity  varies,  but  most  are 
vulnerable.  The  question  is  at  present  clouded  by  the 
statement  that  various  types  of  tubercle  bacilli  exist, 
such  as  the  human  type,  the  bovine  type,  and  the  avian 
type,  and  that  these  types  vary  in  their  power  over  the 
different  animal  species. 

Ignoring  for  the  moment  the  different  types,  tuberculosis 
is  mostly  found  in  cattle  and  pigs.  "  Dogs  and  cats 
occasionally  suffer,  and  monkeys  and  apes  (immune  when 
in  the  wild  state)  are  very  subject  to  it  and  mostly  die  of 
it  in  captivity.  Horses  are  rarely  attacked,  and  sheep 
are  practically  immune.  Of  all  the  home  cattle  slaughtered 
in  the  Glasgow  market  in  1910  (67,849),  12-98  per  cent 
showed  lesions  of  tuberculosis,  and  of  39,724  swine,  6*  12 
per  cent ;  while  out  of  307,784  sheep  not  one  snowed 
tuberculous  lesions.  The  rate  among  cattle  is  excessive 
from  the  large  proportion  of  milch  cows  included,  some 
of  which  are  imported  into  the  city  and  are  kept  in  byres 


NON-SPORING    BACILLI  277 

until  sent  to  be  slaughtered.  Apart  from  that,  however, 
the  figures  suggest  the  influence  of  open-air  life  in 
lowering  the  susceptibility  of  the  sheep  and  the  chances 
of  infection. 

B.  tuberculosis  of  the  human  type  is  pathogenic  for 
guinea-pigs,  less  so  for  rabbits,  and  still  less  so  for 
dogs. 

B.  tuberculosis  of  the  bovine  type  is  very  pathogenic  for 
guinea-pigs,  killing  them  more  quickly  and  producing 
more  extensive  lesions  than  the  human  type ;  while 
intravenous  injection  in  rabbits  causes  an  acute  tuber- 
culosis with  death  in  from  two  to  five  weeks ;  the 
human  type  causing  a  mild,  slow  disease,  usually  lasting 
for  six  months,  and  at  times  failing  to  kill  the  rabbit. 
This  is  the  readiest  method  of  distinguishing  these 
two  types. 

The  human  type  is  found  in  the  majority  of  human 
infections  (46  out  of  60  investigated),  never  in  bovine 
tuberculosis. 

The  bovine  type  is  found  always  in  bovine  tuberculosis, 
and  in  a  considerable  number  of  cases  of  human  tuber- 
culosis (14  out  of  60  sb  23  per  cent),  and  in  all  the  latter 
but  one,  the  lesions  were  of  the  cervical  lymphatic  glands, 
or  were  lesions  of  primary  abdominal  tuberculosis,  that 
is,  were  lesions  which  might  fairly  be  attributed  to  feeding 
or  alimentation.  Moreover  they  were  mostly  in  children, 
so  that  the  deduction  is  made  that  bovine  tuberculosis  is 
transmissible  to  man,  the  milk  of  tuberculous  cows  being 
the  usual  vehicle. 

Bovine  tubercle  bacilli,  in  cultures,  are  shorter,  thicker, 
and  more  regular  in  size  than  the  human  type,  and  their 
growth  on  the  various  media  is  scantier.  They  are  more 
pathogenic  to  cattle  and  swine,  and  less  so  to  man,  which 
may  explain  the  chronicity  of  abdominal  and  cervical 
glandular  tuberculosis. 

A  recent  research  by  Park  and  Krumwiede  confirms  the 
results  of  previous  investigations.  They  state  that  com- 
parative luxuriance  or  sparseness  of  growth  is  in  most 
cases  absolutelv  to  be  relied  on  for  differentiation. 


278         PUBLIC    HEALTH    BACTERIOLOGY 

Their  results  may  be  summarized  thus : — 


Form  of  Tuberculosis 

Pulmonary 

Cer\ 

•ICAL 

Abdominal 

Type  of  bacillus  found 

Human 

Bovine 

Human 

Bovine 

Human 

Bovine 

Ages  of  persons  exam- 
ined : — 
1 6  years  and  "upwards 
5  years  and  up  to  1 6 
Under  5  years    ** 

290 

3 

7 

r  tji 

I 

o1 

O 

13 

9 

I 
12 

8 

14 

6 
6 

3 

6 

IO 

Totals 

300 

I 

36 

21 

26 

19 

The  results  in  606  cases  of  various  kinds  of  tuberculosis, 
investigated  by  different  workers,  are  tabulated  thus 
(three  cases  showed  both  types)  : — 


Total 


Human  Type 


Bovine  Type 


Adults  (16  years  and  upwards) 
Children  (5  to  16  yrs.  inclusive) 
Children  (under  5  years) 


389 

78 

136 


381  -  98  % 
54  =  69  % 
99  *  72  % 


37 


•    2% 
30  % 

27% 


Totals 


603 


534 


% 


The  bacillus  of  avian  tuberculosis  shows  a  more 
luxuriant  and  moister  growth  than  the  human  type,  and 
grows  at  43-5°  C,  is  very  pathogenic  to  rabbits,  scarcely 
pathogenic  to  guinea-pigs,  and  not  at  all  to  dogs,  even 
intravenously,  whereas  the  human  type  produces  an 
acute  infection.  Morphologically  it  is  almost  identical 
with  the  human  type,  and  stains  similarly.  It  is  found 
in  lesions  in  fowls  and  pigeons  and  some  other  bird 
species.  Fowls  fed  on  human  tubercle  bacilli  are  not 
found  to  become  tuberculous.  The  identity  of  the  two 
types  is  said  to  be  established  by  the  experiments  of 
Nocard,  who  rendered  mammalian  tubercle  bacilli 
pathogenic  for  fowls  by  keeping  them  in  the  peritoneal 
cavities  of  hens  in  collodion  sacs  for  six  months.  Con- 
versely, prolonged  cultivation  and  passage  of  the  avian 
type  through  the  mammalian  body  are  said  to  cause  them 
to  approach  closely  to  the  mammalian  type. 

The  bacillus  of  a  tubercle -like  disease  in  a  carp  has 


NON-SPORING    BACILLI  279 

been  called  the  bacillus  of  fish  tuberculosis.  It  grows 
luxuriantly  at  room  temperature  (200  C),  and  does  not  grow 
at  370  C,  its  range  being  15  °  to  300  C.  It  is  non-pathogenic 
for  mammals,  but  kills  frogs  in  a  month.  It  shows  some 
degree  of  acid-fastness. 

Portals  of  Entry. — Inheritance,  ingestion,  inhalation, 
and  inoculation.  Cobbett  recently  traverses  the  theories 
of  Behring,  Calmette,  and  Guerin,  that  the  portal  of  entry 
of  the  tubercle  bacilli,  even  in  the  pulmonary  form,  is  via 
the  alimentary  tract  and  thence  via  the  lymphatics  to  the 
lungs.  He  concludes  that  the  .intestine  is  not  a  common 
port  of  entry  for  the  tubercle  bacilli  which  cause  phthisis. 
(Cobbett,  "  Portals  of  Entry  of  the  Tubercle  Bacillus," 
Jour,  of  Path,  and  Bad.,  vol.  xiv.,  No.  4,  1910,  p.  563). 
Leonard  Findlay  reaches  the  same  conclusion  from 
experiments  on  the  production  of  pulmonary  anthracosis 
in  rabbits  and  guinea-pigs  (Findlay,  "  The  Origin  of  Pul- 
monary Anthracosis,  an  Experimental  Study."  Zeitschrift 
filr  Kinder heilkunde,  1911,  vol.  ii.,  part  2  (June),  p.  293. 
B.M.J.,  1911,  vol.  ii.,  p.  1278.)  Feeding  experiments 
undertaken  for  the  Royal  Commission  on  Tuberculosis 
gave  results  more  in  consonance  with  Calmette' s  theories 
(see  resume  on  page  297  ;  details  in  Appendix  to  Filial 
Report,  vol.  i.,  pp.  48  and  52). 

Toxins. — The  tubercle  bacillus  secretes  no  soluble  toxin 
or  only  in  very  small  amount.  The  chief  toxic  principles 
are  endotoxins  or  bacterial  proteins.  Dead  bacilli  will,  if 
inoculated  in  sufficient  numbers,  produce  tubercle-like 
nodules,  in  which  giant  cells  and  occasionally  caseation  are 
present.  These  results  are  obtained  with  intravenous  and 
intraperitoneal  injections,  whereas  subcutaneous  injection 
produces  a  sterile  abscess  (cold  abscess). 

The  hope  of  producing  an  active  immunity  led  Koch  to 
employ  various  means  to  extract  from  dead  and  living 
bacilli  the  complex  bodies  bound  in  them. 

1.  Original  Tuberculin,  T.A.  (Koch  1890-91). — Tubercle 
bacilli  are  grown  in  5  per  cent  glycerin  broth  for  six 
to  eight  weeks.  The  entire  culture  is  then  heated  on 
the  water-bath  at  8o°  C.  until  reduced  to  one-tenth  of 
its  original  bulk.  It  is  then  filtered  through  sterile  filter- 
paper  or  through  porcelain  filters.     The  filtrate  is  a  thick, 


280         PUBLIC    HEALTH    BACTERIOLOGY 

brown,  syrupy  liquid,,  containing  the  products  of  growth 
in  the  culture  medium  and  a  50  per  cent  extract  of  the 
bodies  of  the  bacilli  by  glycerin  ;  all  in  so  far  as  these  are 
indestructible  by  heat.  Stored  in  a  cool  dark  place  it 
keeps  indefinitely,  the  glycerin  acting  as  a  preservative. 
The  dose  of  this  preparation  used  in  cattle  is  0-25  c.c,  but 
the  stock  is  usually  diluted  with  0-5  per  cent  carbolic  water 
four  times,  making  the  dose  2  c.c.  Such  a  dose  in  a 
healthy  man  causes  in  three  to  four  hours  malaise,  tendency 
to  cough,  laboured  breathing,  and  moderate  pyrexia,  all 
passing  off  in  twenty-four  hours.  In  a  man  suffering 
from  tuberculosis,  such  a  dose  would  give  rise  to  such  an 
excessive  reaction  as  probably  to  cause  death.  Even 
o-oi  c.c.  causes  all  the  above  symptoms  in  an  aggravated 
degree,  together  with  marked  inflammatory  reaction 
around  any  tuberculous  focus,  resulting  in  necrosis  but 
not  killing  the  bacilli.  This  is  now  known  as  the  "  tuber- 
culin reaction,"  and  is  much  used  in  veterinary  practice. 
It  is  also  used  in  clinical  work  for  help  in  diagnosis  of  obscure 
affections  suspected  to  be  tuberculous. 

i.  Subcutaneously  :  Use  diluted  tuberculin,  1-1000,  so 
that  1  c.c.  =  1  mgr.  of  tuberculin.  Koch  in  1890  used 
1  «mgr.,  but  now  most  clinicians  begin  with  o-i  mgr.  or 
0-2  mgr.  If  no  reaction  occurs,  after  three  to  four  days 
give  1  mgr.  If  again  no  reaction  follows,  wait  three  to 
four  days  and  try  5  mgr.,  and  finally,  if  still  negative, 
10  mgr.,  but  no  more.  If  still  no  reaction,  the  person  may 
be  considered  tubercle-free.  Take  temperature  regularly 
before  and  after  test. 

ii.  Cutaneous  test  (von  Pirquet,  1907).  For  this  test 
von  Pirquet  first  suggested  a  25  per  cent  solution  of  "  old 
tuberculin,"  but  the  latter  is  now  used  undiluted.  The 
patient's  skin  on  the  flexor  surface  of  the  forearm  is 
sterilized.  Two  separate  drops  of  the  tuberculin  are  placed 
on  the  skin,  2  to  4  in.  apart.  With  a  small  metal  bore 
the  skin  is  scarified  at  a  point  midway  between  the  two 
drops,  and  then  that  is  covered  by  the  drops.  Within 
twenty-four  to  forty-eight  hours  in  tuberculous  patients 
redness  round  the  points,  a  papule  over  the  scarified  surface, 
and  minute  vesicles  all  appear.  The  control  area  shows 
a  slight  traumatic  redness  which  soon  passes  off.      In  a 


NON-SPORING    BACILLI  281 

negative  result  all  three  areas  show  this  slight  reaction. 
Intermediate  reactions,  in  all  degrees,  are  observed. 
70  per  cent  of  adults  give  a  positive  result,  probably  from 
healed  tuberculosis. 

iii.  Inunction  of  lanolin,  containing  50  per  cent  of 
tuberculin  (Moro's  test). 

iv.  Ophthalmo-tuberculin  reaction  (1907,  Wolff-Eisner  ; 
Calmette).  For  this  test,  "old  tuberculin"  is  treated 
with  95  per  cent  alcohol,  and  the  precipitate  dissolved  in 
water,  and  re-precipitated  and  dissolved  several  times, 
being  finally  made  up  to  a  1  per  cent  watery  solution.  One 
drop  of  this  is  instilled  into  the  conjunctival  sac  and 
allowed  to  spread  all  over  it.  A  sharp  congestion  of  both 
the  ocular  and  palpebral  conjunctivae  results  in  six  to  ten 
hours  in  the  case  of  a  positive  reaction,  and  passes  off  in 
twenty-four  to  thirty-six  hours.  In  children,  half  the 
dose  is  used  and  the  acme  is  reached  sooner.  In  several 
cases  the  results  of  this  test  have  been  disastrous,  the 
excessive  reaction  leading  to  destruction  of  tissue  and 
blindness  in  the  eye  used  for  the  test. 

2.  New  Tuberculin  (Koch,  1897) — Was  introduced  to  pro- 
duce anti-bacterial  immunity.  It  was  prepared  thus :  bacilli 
from  young  virulent  cultures  were  ground  in  an  agate  mill, 
washed  with  distilled  water,  and  centrifugalized.  The 
clear  fluid  was  decanted  and  called  tuberculin-O.  The 
deposit  was  dried,  ground,  and  treated  as  before  ;  and 
this  was  repeated  several  times  until  all  the  residue  went 
into  emulsion ;  the  resulting  fluids  mixed  together  are 
tuberculin-R.  Tuberculin-0  gives  no  precipitate  with 
glycerin  added  to  make  a  50  per  cent  solution.  Tuber- 
culin-R does  precipitate  in  50  per  cent  glycerin.  Tuber- 
culin-0  gives  a  reaction  on  injection  more  readily  than 
tuberculin-R. 

3.  Bacillary  Emulsion  (Koch,  1901). — This  is  a  1  per 
cent  emulsion  of  pulverized  bacilli  in  distilled  water, 
allowed  to  sediment  for  several  days,  and  then  the  super- 
natant liquid  is  mixed  with  an  equal  bulk  of  glycerin, 
making  the  whole  50  per  cent.  1  c.c.  =  5  mgr.  solids. 
It  resembles  a  mixture  of  tuberculin-0  and  tuberculin-R. 

4.  Bouillon  Filtre  (Denys,  1905). — A  culture  of  tubercle 
bacilli    in    5    per    cent    glycerin    broth,    filtered    through 


282         PUBLIC     HEALTH    BACTERIOLOGY 

Chamberland  filters  and  not  heated,  but  0-25  per  cent 
phenol  added  to  preserve.  Corresponds  to  "  old 
tuberculin,"  unconcentrated  and  unheated,  and  therefore 
supposed  by  Denys  to  contain  important  soluble  but 
thermolabile  substances. 

Therapeutic  uses  of  the  Tuberculins.  —  (So-called 
Vaccine  Therapy.) — For  these  purposes,  tuberculin-R  and 
bacillary  emulsion  are  mostly  used  in  doses  beginning  at 
o-ooi  mgr.  (yyVo)'  and  gradually  increased  so  long  as  the 
dose  causes  no  greater  disturbance  of  temperature  than 
0-5°  F.  In  Wright's  method  of  treatment  the  initial  dose 
is  usually  ToVo  mgr->  and  is  rarely  increased  beyond  ToVtt 
mgr.  The  dose  is  administered  after  determination 
of  the  opsonic  index  of  the  patient,  and  subsequent  doses 
are  only  administered  during  the  positive  phase  of  the 
reaction  to  the  previous  dose.  To  determine  this, 
repeated  observations  of  the  opsonic  index  have  to  be 
made.  As  a  result  of  experience  gained  by  a  large  number 
of  observations,  some  authorities  advise  the  use  of  smaller 
doses,  5l[iM  mgr.  every  ten  days,  gradually  increased  to 
40*00  mgr.  in  six  months'  time,  without  opsonic  index 
estimation,  but  simply  watching  the  clinical  symptoms. 

In  the  serum  of  patients  so  treated  there  is  evidence  of 
the  formation  of  bodies  antagonistic  to  tuberculin,  of  the 
nature  of  immune-bodies,  precipitins,  and  opsonins. 

Antituberculous  Sera. —  Maragliano's  Serum. —  Made 
by  him  by  immunizing  dogs,  asses,  and  horses,  by  injecting 
a  mixture  of  the  filtrate  of  an  unheated  broth  culture 
(1  part)  and  an  aqueous  bacillary  extract  at  ioo°  C.  (3 
parts).  The  animal  is  bled  after  four  to  six  months. 
Of  the  serum,  2  c.c.  are  injected  subcutaneously  every  two 
days.  Improvement  has  been  noted  in  non-febrile  cases. 
The  serum  is  capable  of  protecting  an  otherwise  healthy 
animal  against  a  fatal  dose  of  tuberculin. 

Marmorek's  Serum. — Marmorek  believes  the  tubercle 
bacillus  does  not  produce  in  ordinary  media  the  same 
toxins  that  it  does  in  the  body,  where  it  has  to  resist  the 
antagonism  of  the  body  cells.  To  combat  this  he  first 
grows  the  bacilli  on  a  leucotoxic  serum  (produced  by 
inoculating  calves  with  guinea-pig  leucocytes),  and  then 
on   a  medium   containing  liver   extract,   the  liver  being 


NON-SPORING    BACILLI  283 

regarded  as  the  most  antituberculous  tissue  in  the  body. 
He  thereafter  uses  these  bacilli  (which  he  states  yield  no 
tuberculin)  to  immunize  animals,  and  for  the  serum 
produced  he  claims  high  curative  powers. 

Tuberculin  Tests  applied  to  Cattle. — In  cattle, 
tuberculosis  may  be  present  without  any  very  apparent 
symptoms  until  an  advanced  stage  is  reached.  Routine 
examination  of  herds  by  the  tuberculin  test  has  therefore 
become  one  of  the  necessary  measures  in  public  sanitation, 
in  order  that  the  milk  of  tuberculous  cows  may  be  excluded 
from  consumption,  and  that  such  cows  may  be  eliminated 
from  herds  (Bang's  System).  Mohler  states  that  an 
accurate  diagnosis  is  established  in  97  per  cent  of  the  cases. 
Stall  the  animal  and  take  the  temperature  in  the  rectum 
every  two  hours  from  6  a.m.  until  midnight.  Make  the 
injection  then  (subcutaneously).  Begin  to  take  the  tem- 
perature at  6  a.m.  and  continue  as  on  the  preceding  day. 
The  dose  is  usually  0-25  c.c.  of  old  tuberculin.  A  positive 
reaction  consists  in  febrile  and  constitutional  signs,  with 
marked  congestion  around  any  focus  of  tuberculosis.  The 
febrile  reaction  begins  six  to  ten  hours  after  injection, 
reaches  its  height  in  nine  to  fifteen  hours,  and  declines 
to  normal  in  eighteen  to  twenty-six  hours.  A  rise  of 
20  F.  or  more  above  the  maximum  of  the  previous  day 
should  be  regarded  as  a  positive  reaction.  In  a  doubtful 
case,  repeat  in  four  to  six  weeks.  (Normal  rectal  tempera- 
ture, ioo°  to  1020  F.  ;  normal  pulse,  45  to^55  beats  per 
minute. 

Methods  of  Detection. — 1.  Microscopic. — In  sputum: 
select  a  yellowish  piece,  make  a  film,  and  stain  by  acid-fast 
method.  If  not  found,  make  solution  of  sputum  by 
gradual  addition  of  NaOH  solution,  boiling  the  while  ; 
then  sediment  or  centrifuge.  Or  mix  in  5  per  cent  carbolic 
or  2  per  cent  lysol,  and  stand ;  gradual  solution,  and 
tubercle  bacilli  precipitated.  Or  add  antiformin  (or  a 
mixture  of  equal  parts  of  solutions  of  NaCIO  and  NaOH, 
each  7-5  per  cent)  ;  dissolves  in  a  few  minutes.  If  a  20 
per  cent  dilution  of  antiformin  is  used,  the  tubercle  bacilli 
are  not  damaged,  and  all  the  other  bacteria  are  killed. 
Centrifuge  or  sediment ;  wash  twice  with  normal  salt 
solution,  and  sediment  can  then  be  used  to  make  culture, 


284         PUBLIC    HEALTH    BACTERIOLOGY 

or  inoculate  guinea-pig.  In  urine  :  sediment  or  centri- 
fuge, make  films,  and  examine.  To  avoid  smegma  bacilli, 
take  specimen  after  cleansing  meatus,  or  by  catheter, 
and  in  staining  decolorize  with  absolute  alcohol  after 
the  acid  (see  pp.  163  and  285.  In  pus,  faeces,  and  tissues  : 
dissolve  in  antiformin  as  above,  and  examine  deposit.  In 
milk :  centrifuge,  take  sediment  and  fat,  and  mix,  make 
films,  fix,  remove  fat  with  ether  (not  absolutely  necessary), 
stain,  and  examine.  Also  inject  o-i  c.c,  1  c.c.  and  3  c.c.  of 
mixed  sediment  and  fat  into  three  guinea-pigs  respectively ; 
kill  in  three  weeks  and  examine  peritoneum,  post-sternal 
glands,  pancreas,  spleen,  and  liver. 

2.  Inoculation. — Select  a  guinea-pig  and  inject,  sub- 
cut  aneously  or  intraperitoneally,  the  fluid  to  be  tested,  or 
the  washed  sediment  from  an  antiformin  solution  of  tissues, 
faeces,  or  sputum.  The  animal  usually  dies  in  six  weeks, 
if  tubercle  bacilli  are  in  the  injected  substance,  with  local 
and  glandular  changes,  the  spleen  showing  numerous 
tuberculous  nodules  and  being  swollen  as  a  whole. 

3.  Cultivation — Is  made  from  the  inoculated  animal,  or 
from  antiformin  solution  sediments,  or  from  sputum 
treated  with  2  per  cent  ericolin. 

4.  Tuberculin  Reactions. 

OTHER     ACID-FAST     BACILLI. 

Besides  the  bacilli  of  human,  bovine,  avian,  and  fish 
tuberculosis,  there  are  other  bacteria  which  are  acid- 
fast,  as  already  enumerated  on  page  163.  Such  are : 
Moeller's  Timothy  -  grass  bacillus  I  (from  infusions  of 
Timothy  grass) ,  Moeller's  Timothy-grass  bacillus  II  (from 
the  dust  of  a  hay-loft),  butter  bacilli  (isolated  from  butter 
by  Petri,  Rabinowitch,  Korn,  Tobler,  Coggi,  and  others), 
mistbacillus  (from  dung  by  Moeller) .  All  these  are  mor- 
phologically very  similar  to  the  tubercle  bacilli,  are  ex- 
tremely acid-fast,  and  produce  lesions  in  guinea-pigs  on 
injection,  which  closely  resemble  tubercles.  They  are, 
however,  easily  distinguished  by  their  rapid  growth  on 
ordinary  media,  colonies  being  visible  in  twenty-four  hours 
at  370  C.  (in  the  case  of  the  tubercle  bacillus,  the  earliest 
is  eight  days),  and  by  their  growth  in  most  instances  at 


NON-SPORING    BACILLI  285 

room  temperature.  The  cultures  themselves  are,  however, 
similar  to  those  of  tubercle  bacilli,  and  of  one  another. 

Johne's  Bacillus  is  the  bacillus  of  "  chronic  bovine 
pseudo-tuberculous  enteritis,"  a  disease  characterized  by 
corrugated  thickenings  of  the  mucous  membrane  of  the 
small  intestine  (especially),  the  bacilli  occurring  in  large 
numbers  in  the  lesions  and  in  scrapings  from  the  surface. 
The  bacilli  are  like  the  tubercle  bacilli,  but  slightly  shorter  ; 
they  are  equally  acid-fast.  They  have  not  yet  been 
cultivated  on  artificial  media. 

Bacillus  Smegmatis  (the  smegma  bacillus)  occurs  in 
large  numbers  in  the  preputial  secretions  of  the  male,  the 
external  genitals  of  the  female,  and  within  the  folds  of  the 
thighs  and  buttocks.  The  bacilli  are  usually  found  in 
clumps  on  the  mucous  membrane,  and  occasionally  in  the 
superficial  layers  of  the  epithelium,  both  inside  and  outside 
the  cells.  They  were  first  described  by  Lustgarten  in  1884, 
who  found  them  in  a  number  of  syphilitic  lesions,  and  who 
thereupon  believed  them  to  be  the  cause  of  that  disease. 
Further  work  by  Alvarez  and  Tavel,  Klemperer,  and 
others,  showed  that  they  were  harmless  saprophytes. 
They  are  very  similar  to  the  tubercle  bacillus,  but  are 
more  varied  in  size  (usually  distinctly  shorter),  at  times 
slightly  curved  and  short.  They  are  not  easily  stained, 
and  once  stained  resist  decolorization  by  acids,  but  not  so 
strongly  as  tubercle  bacilli.  They  are  said  to  give  up  the 
stain  to  absolute  alcohol,  but  contradictory  statements, 
are  made.  On  this  basis  is  founded  Pappenheim's  method 
of  staining,  where  a  film  stained  with  hot  carbol-fuchsin 
is  treated  with  absolute  alcohol  containing  1  per  cent 
rosolic  acid  (corallin),  methylene-blue  to  saturation,  and 
16  per  cent  of  glycerin.  The  tubercle  bacilli  are  red, 
the  bacilli  smegmatis  blue.  They  are  cultivated  with 
great  difficulty,  and  first  on  serum  or  ascitic  media.  They 
are  non-pathogenic,  so  far  as  tested.  Their  growth  on 
media  is  slow  (five  to  six  days)  ;  the  colonies  are  yellowish- 
white,  and  corrugated  like  tubercle  bacillus  colonies. 
Bacilli  of  the  smegma  group  have  occasionally  been 
demonstrated  in  sputum  and  in  secretions  from  the  throat 
and  tonsillar  crypts. 

Bacillus   Leprae.  —  A  bacillus  closely  resembling  the 


286         PUBLIC    HEALTH    BACTERIOLOGY 

tubercle  bacillus  in  size  and  in  acid-fastness.  These  bacilli 
usually  stain  uniformly,  not  showing  beading.  They  are 
non-motile,  non-flagellar,  and  non-sporing.  They  have 
not  been  successfully  cultivated,  and  attempts  to  inoculate 
animals  have  failed.  They  are  readily  stained  by  Gram's 
method.  They  are  found  in  large  numbers  in  the  cutaneous 
lesions  of  tubercular  leprosy,  and  occur  for  the  most  part 
within  the  protoplasm  of  the  round  granulation  tissue 
cells.  They  are  also  found  in  the  lymphatic  glands,  and 
in  smaller  numbers  in  the  liver  and  spleen.  The  spread  of 
the  disease  is  by  the  lymphatics.  The  earliest  lesion  is 
usually  a  nasal  ulcer  at  the  junction  of  the  bony  and 
cartilaginous  septum.  In  the  anaesthetic  form,  or  nerve 
leprosy,  the  bacilli  are  found  in  the  diffuse  infiltrations  in 
the  nerves,  rarely  in  the  trophic  lesions  resulting.  Lepers 
react  to  tuberculin,  and  50  per  cent  are  said  to  give  the 
Wassermann  reaction.  The  nasal  mucus  and  saliva  (in  a 
less  degree)  are  the  vehicles  by  which  the  disease  is  spread. 
^Diagnosis. — Animal  inoculation  is  negative. 

ACTINOMYCOSIS,  OR^THE^RAY  FUNGUS  DISEASE. 

Actinomycosis  is  a  disease  of  cattle  and  man.  [It 
occasionally  affects  sheep,  dogs,  cats,  and  horses.  Its 
usual  sites  are  the  regions  of  the  face,  mouth,  and  pharynx. 
In  cattle,  the  lower  jaw  is  most  frequently  affected,  the 
disease  taking  the  form  of  tumour  formation,  the  so-called 
"  lumpy  jaw."  The  tumours  are  often  nodulated  and 
consist  of  fibrous  tissue  with  irregular  abscess  cavities 
throughout.  When  an  abscess  discharges,  the  pus  is  of 
a  yellowish-green  colour,  slimy  in  character,  and  contains 
small  granular  bodies,  visible  to  the  eye  and  distinctly 
palpable,  and  of  a  pale  sulphur  colour.  These  granules 
are  found  on  examination  to  be  composed  of  rosette-like 
masses  of  the  fungus  actinomyces,  or  ray  fungus,  first 
described  by  Boellinger  in  1877.  I*  *s  now  classed  among 
the  trichomycetes  or  higher  bacteria,  and  by  some  as  a 
true  mould ;  that  is,  forms  composed  of  threads  which 
show  true  branching  and  multiply  by  spore-shaped  bodies, 
which  usually  appear  in  chains — the  gonidia  or  spores. 
(Madura  foot,  or  mycetoma,  is  similar  in  nature  to  actino- 
mycosis.) 


NON-SPORING    BACILLI  287 

Description. — An  anaerobe  of  slow  growth,  growing  best 
at  370  C. ;  and  in  a  shake  culture  in  glucose  agar,  the 
colonies  are  most  numerous  5  to  10  mm.  below  the  surface 
of  the  medium,  the  inference  being  that  a  trace  of  oxygen 
is  an  advantage.  The  colonies  are  round,  dense,  and 
greyish-white  in  colour  (chalky)  ;  sometimes  they  are 
rosette-shaped.  Another  variety  has  been  described  which 
is  an  aerobe,  growing  in  three  to  four  days  to  little 
transparent  drops,  becoming  later  amber,  and  then  reddish- 
yellow  in  colour.  This  variety  has  been  grown  on  gelatin, 
which  it  liquefies.  In  cultures,  club-shaped  forms  have 
not  been  found  in  the  aerobic  variety,  but  have  been  noted 
by  J.  H.  Wright  in  the  anaerobic  variety  when  grown  in 
the  presence  of  serum  or  other  animal  fluids.  It  is  believed 
that  several  kinds  have  been  described  under  the  one 
name,  and  that  further  research  is  needed  to  differentiate 
these.  The  anaerobic  form  grows  in  broth,  forming  heavy 
flocculent  masses  (solid  white  mulberry  granules)  at  the 
bottom  of  the  tube  ;  no  clouding  nor  surface  growth. 

The  fungus  is  described  as  having  three  forms : 
(1)  Filaments,  more  or  less  radially  arranged,  0-5  micron 
thick,  and  closely  interlaced.  These  form  the  central  core 
of  the  colony.  (2)  At  the  periphery,  refringent  club-shaped 
bodies,  structureless  and  homogeneous ;  whereas  the 
filaments  show  a  sheath,  enclosing  a  granular  protoplasm. 
(3)  Spores  or  gonidia  are  coccus-like  bodies,  found  between 
the  filaments  of  the  central  mass ;  are  variously  regarded 
as  real  gonidia,  or  as  degeneration  products,  or  con- 
taminating cocci.  In  cultures,  gonidia  are  developed  at 
the  ends  of  the  filaments,  and  such  gonidia  have  a  higher 
resistance  to  heat  than  the  simple  filaments,  half  an  hour 
at  750  C.  being  required  to  kill  spore-bearing  cultures,  and 
the  same  time  at  650  C.  for  spore-free  cultures.  The 
filaments  are  Gram-positive  and  acid-fast. 

Pathogenicity. — In  man,  the  disease  tends  to  generalize  ; 
in  the  ox,  to  remain  local.  The  point  of  entrance  in  man 
is  usually  by  a  carious  tooth,  by  the  tonsil,  or  by  some 
abrasion.  The  corresponding  glands  are  next  affected, 
and  later  metastatic  abscesses  are  formed  in  the  skin  and 
elsewhere.  The  symptoms  resemble  those  of  chronic 
tuberculosis,  for  which  the  patient  is  usually  treated.     The 


288         PUBLIC    HEALTH    BACTERIOLOGY 

disease  is  acquired  probably  from  hay,  straw,  and  grain  r 
and  possibly  by  milk  of  infected  cattle.  An  actinomyces 
has  been  isolated  from  hay  and  straw,  and  in  cattle,  grains 
have  been  found  embedded  in  the  centre  of  growths. 
Inoculation  of  the  ox  has  produced  the  disease  ;  in  the 
smaller  animals,  characteristic  colonies  and  lesions  may 
follow,  but  little  growth. 

Isolation. — May  be  easy  or  very  difficult.  The  pus  is 
washed  in  salt  solution  and  sown  in  melted  glucose  agar. 
If  much  contamination  is  found,  keep  washed  granules 
for  several  weeks  in  a  dry  state,  and  try  again. 


Summary  of  the  Final  Report  of  the  British  Royal 
Commission  on  Tuberculosis  Issued  in  June,  1911. 

The  British  Royal  Commission  appointed  in  1901  to 
inquire  into  the  relations  of  human  and  animal  tuber- 
culosis, issued  its  final  report  in  June,  191 1.  The 
Commission  was  appointed  on  account  of  the  diversity 
of  opinion  which  was  manifested  at  the  International 
Congress  on  Tuberculosis,  held  in  London  in  1901,  when 
the  statement  was  made  by  Koch  that  human  tuberculosis 
cannot  be  transmitted  to  cattle,  and  that  bovine  tubercu- 
losis is  not  dangerous  to  man.  The  results  of  the  work  of 
the  Commission,  and  of  much  other  parallel  work,  are  to 
traverse  directly  both  statements.  The  Final  Report, 
extends  to  about  fifty  pages  (there  are  7  volumes  of  an 
Appendix),  and  may  be  usefully  summarized  thus  : — 

The  report  is  unanimous.  It  is  based  on  the  isolation 
of  the  bacilli  from  the  lesions  of  the  natural  disease  ;  the 
investigation  of  the  cultural  characters  of  the  bacilli 
isolated,  and  the  study  of  their  effects  when  introduced  in 
varying  doses  and  by  several  methods  into  different 
animals.  The  species  of  animals  used  have  been  cattle, 
rabbits,  guinea-pigs,  pigs,  goats,  chimpanzees,  monkeys, 
horses,  rats,  mice,  dogs,  cats,  and  birds. 

The  experimental  methods  of  infection  used  were : 
subcutaneous,  intravenous,  intraperitoneal,  and  by  feeding 
(oral).  Inhalation  was  not  tried.  The  findings  are  based 
on  the  researches  of  their  own  staff. 


NON-SPORING    BACILLI  289 

Three  types  of  tubercle  bacilli  are  described  : — 
(i)  Bovine   tubercle    bacilli  —  the    only  kind    found    in 
natural  tuberculosis  of  cattle. 

(2)  Human  tubercle  bacilli — the  kind  most  commonly 
found  in  man,  but  not  the  only  kind  so  found. 

(3)  Avian  tubercle  bacilli  —  the  only  kind  found  in 
natural  tuberculosis  of  birds. 

1.  Bovine  tubercle  bacillus  is  taken  as  the  standard 
for  comparison. 

Summary  of  its  Characters.— (a).  Cultural. — Grows 
slowly  on  serum,  and  at  the  end  of  two  to  three  weeks 
shows  on  surface  as  a  thin,  greyish,  uniform  growth,  not 
wrinkled  nor  pigmented.  According  to  the  rate  and 
luxuriance  of  growth  on  glycerin  media,  bovine  tubercle 
bacilli  may  be  divided  into  three  grades.  Nevertheless  it  is 
insisted  that  rate  and  kind  of  growth  should  not  be  the 
sole  basis  of  identification  as  bovine  type,  but  only  when 
considered  along  with  results  of  inoculation  experiments. 

(b).  Effects  on  animals. — Produces  characteristic  effects 
when  inoculated  into  calves  and  rabbits  in  certain  doses. 

Calves :  subcutaneous  injection  in  neck  of  50  mgr.  of 
culture  under  three  weeks  old.  causes  severe  general 
tuberculosis,  starting  at  point  of  inoculation;  and  death 
usually  within  eight  weeks. 

Rabbits  :  intravenous  injection  of  o-oi  mgr.  or  o-i  mgr. 
of  culture  causes  generalized  miliary  tuberculosis,  ending 
in  death  within  5  weeks  ;  intraperitoneal  injection  of  o*i 
mgr. — death  in  13  to  48  days;  intraperitoneal  injection 
of  i-o  mgr. — death  in  10  to  38  days;  subcutaneous  injection 
of  1  mgr. — death  in  29  to  165  days;  subcutaneous  injection 
of  10  mgr. — death  in  28  to  101  days. 

These  results  are  very  striking  and  definite,  and  along 
with  cultural  tests  afford  a  trustworthy  means  of  .recog- 
nizing bovine  tubercle  bacilli.  Later  it  was  considered 
sufficient  to  inoculate  rabbits,  as  results  are  very  reliable. 

(c).  Other  properties. — Subcutaneous  inoculation  in  very 
small  doses  invariably  produces  acute  tuberculosis  in  the 
chimpanzee,  monkey,  and  guinea-pig. 

In  the  goat,  the  pig,  and  the  cat,  general  tuberculosis 
is  readily  induced. 

The  rat  and  the  mouse   are   highly  resistant  to  sub- 

19 


290         PUBLIC    HEALTH    BACTERIOLOGY 

cutaneous  inoculation  ;  intraperitoneally,  the  bacilli  mul- 
tiply in  the  body  but  do  not  produce  tubercles. 

Dogs  are  highly  resistant  to  subcutaneous  inoculation, 
but  succumb  to  general  tuberculosis  when  large  doses  are 
given  intravenously  or  intraperitoneally. 

In  the  fowl,  intravenous  injection  of  bovine  tubercle 
bacilli  caused  death  in  50  per  cent,  with  wasting,  cedema 
of  lung,  and  pallor  of  liver.  In  a  few,  definite  tubercles 
were  found  in  the  lungs  and  minute  necrotic  areas  in  the 
liver.  Death  is  apparently  due  to  toxaemia,  as  dead  bacilli 
have  the  same  effects.  Intraperitoneally  and  intramuscu- 
larly, even  in  large  doses,  only  local  lesions  are  produced, 
and  there  is  no  dissemination. 

Horses :  subcutaneously  or  orally,  no  progressive 
tuberculosis  is  produced.  Intravenously,  10  mgr.  cause 
death  from  acute  tuberculosis  in  twenty  days. 

(d).  Stability  in  culture. — Subcultured  for  long  periods, 
(one  case,  1487  days  =  4  years),  no  great  loss  of  virulence 
was  found. 

2.  Human  tubercle  bacillus. — The  human  type  is  taken 
as  that  bacillus  which  has  been  found  in  the  majority  of 
cases  of  human  tuberculosis.  Its  chief  characters  are  : 
on  serum,  it  grows  more  rapidly  than  the  bovine  type, 
hence  it  is  called  "  eugonic,"  as  opposed  to  "  dysgonic," 
the  term  applied  to  the  bovine  bacillus.  On  glycerin 
media,  the  growth  tends  to  become  wrinkled ;  and  on 
all  media  becomes  pigmented  to  a  greater  or  less  extent. 
Its  effects  on  animals  place  it  in  still  greater  contrast 
to  the  bovine  type. 

Calves :  subcutaneous  injection  in  neck  of  50  mgr. 
of  culture  under  three  weeks  old,  does  not  produce  pro- 
gressive tuberculosis,  nor  does  it  kill.  Only  a  local  lesion 
results,  which  later  becomes  fibrous.  In  about  half  the 
cases,  the  infection  did  not  extend  beyond  the  nearest  glands. 

Rabbits  :  intravenous  injection  of  o-i  mgr.  to  1  mgr. 
of  culture  causes  slowly  progressive  tuberculosis  with 
limited  lesions,  and  death  (for  the  majority)  after  three 
months  (thirteen  weeks).  Intraperitoneally,  1  mgr.  ; 
animal  alive  after  three  months.  Subcutaneous  injection 
of  1  to  100  mgr.  ;  animals  survived  or  were  killed  in 
94  to  725  days.     In  certain  cases  1  mgr.  or  o-i  mgr.  intra- 


NON-SPORING    BACILLI  291 

venously  acted  like  the  bovine  type,  killing  with  acute  and 
rapid  tuberculosis ;  in  these  cases,  however,  o-oi  mgr.  never 
killed  within  three  months,  thus  easily  distinguishing  the 
bacilli  from  those  taken  as  the  standard.  Hence  this  dose 
is  the  best  to  use  intravenously. 

Effects  on  chimpanzee  and  monkey  in  producing  acute 
tuberculosis,  are  similar  to  those  produced  by  like  doses  of 
the  bovine  tubercle  bacillus. 

In  guinea-pigs,  it  produces  acute  tuberculosis,  but  dura- 
tion of  life  is  longer  than  with  the  same  dose  of  bovine 
tubercle  bacilli. 

In  the  goat,  pig,  and  cat,  great  resistance  is  found,  only 
slight  retrogressive  lesions  being  produced. 

In  the  dog,  the  effects  are  similar  to  the  bovine  tubercle 
bacillus,  that  is,  there  is  great  resistance  to  subcutaneous 
injection,  but  large  doses  given  intravenously  or  intra- 
peritoneally  cause  generalized  tuberculosis  and  death. 

In  the  fowl,  the  effects  are  the  same  as  those  produced 
by  the  bovine  tubercle  bacillus. 

In  a  horse,  subcutaneous  injection  of  50  mgr.  produced 
only  local  disease. 

The  human  tubercle  bacillus  has  not  shown  any  alteration 
in  cultural  characters  on  prolonged  cultivation. 

Resume. — The  human  tubercle  bacillus  is  distinguished 
from  the  bovine  tubercle  bacillus  by  (1)  Its  more  ready 
growth  on  artificial  media  ;  and  (2)  The  results  of  inocula- 
tion into  rabbits,  calves,  cats,  pigs,  and  goats.  They 
are  alike  in  that  they  readily  produce  tuberculosis  in 
chimpanzees,  monkeys,  and  guinea-pigs,  and  in  that 
the  lesions  produced  in  these  animals  are  the  same  in 
distribution  and  structure. 

3.  Avian  tubercle  bacillus. — The  avian  tubercle  bacillus 
forms  a  slimy,  whitish  growth,  easily  emulsified  (difference 
from  human  and  bovine).  It  grows  badly  on  serum,  but 
especially  well  on  glycerinated  media.  Inoculation  into 
animals  produces  effects  markedly  contrasting  with  those 
given  by  bovine  and  human  tubercle  bacilli. 

Fowls  are  very  susceptible  to  intravenous,  intramuscular, 
and  subcutaneous  inoculation  of  the  avian  tubercle  bacilli, 
and  also  to  feeding.  In  the  former  modes,  the  lesions  are 
in  the  spleen  and  liver,  and  frequently  in  the  lungs,  cervical 


292         PUBLIC    HEALTH    BACTERIOLOGY 

glands,  muscles,  and  bones.  After  the  feeding  method, 
there  are  similar  lesions,  with,  in  addition,  characteristic 
tuberculous  lesions  in  the  mucous  membrane  of  the 
intestines. 

Parrots  show  similar  results,  but  by  feeding  method 
do  not  so  regularly  show  intestinal  lesions.  Parrots  are 
susceptible  to  both  the  bovine  and  human  tubercle  bacilli, 
by  inoculation  and  feeding  ;  the  effects  are  similar  to  those 
produced  by  the  avian  type,  except  that  the  bovine  type 
is  apparently  the  most  virulent  for  parrots. 

The  rabbit  and  the  mouse  are  the  only  two  mammals 
in  which  the  avian  tubercle  bacillus  causes  progressive 
tuberculosis. 

Rabbits  :  moderately  large  doses,  by  inoculation, 
produce  a  fatal  issue  ;  the  bacillus  is  less  virulent  than 
bovine  tubercle  bacillus,  but  more  virulent  than  human 
tubercle  bacillus.  The  distribution  of  the  lesions  differs 
most  markedly  from  that  set  up  by  the  bovine  and  human 
types.  Intravenously,  I  to  10  mgr.  lead  to  speedy  death, 
with  great  multiplication  of  the  bacilli  in  the  organs,  which 
show  general  pallor  ;  slight  oedema  of  the  lungs,  slight 
enlargement  (with  tubercles)  of  the  spleen.  If  the  animal 
lives  four  to  five  weeks,  the  spleen  is  greatly  enlarged  owing 
to  formation  of  tubercles,  and  tubercles  are  found  in  the 
liver  and  to  a  less  extent  in  the  lungs.  In  o-ooi  mgr.  dose, 
the  disease  is  very  chronic,  and  resembles  that  produced 
by  subcutaneous  injection,  the  joints  being  affected.  Sub- 
cutaneous injection  of  doses  of  50  mgr.  down  to  a  fraction 
of  1  mgr.  causes  very  chronic  disease,  and  the  lesions  have 
the  same  distribution,  irrespective  of  size  of  dose  :  local 
lesion  and  nearest  lymphatic  glands ;  liver  and  spleen 
rarely  affected  ;  kidneys  vary  ;  but  most  commonly  and 
characteristically  there  is  a  tuberculosis  of  the  joints  of 
the  limbs,  which  runs  a  chronic  course.  Joint  tuberculosis 
occasionally  follows  intravenous  injection  of  human 
tubercle  bacillus,  but  has  not  been  observed  after  sub- 
cutaneous injection  of  the  rabbit  with  the  human  virus. 
It  has  however  been  noted  after  subcutaneous  injection 
of  the  bovine  type,  when  the  animal  survives  for  a  long 
period,  i.e.,  the  disease  is  chronic.  By  feeding,  similar 
lesions  are  produced,  with  local  intestinal  ones. 


NON-SPORING    BACILLI  293 

Mouse :  General  tuberculosis  by  subcutaneous  or 
intraperitoneal  injection,  and  by  feeding. 

Calf,  pig,  goat,  monkey,  guinea-pig,  horse,  cat,  and  rat : 
all  behave  alike  to  the  avian  type.  It  never  produces  a 
progressive  tuberculosis,  but  may  kill  them  in  a  large  dose 
given  intravenously. 

Dogs  :   are  immune  intravenously. 

Chimpanzee :  injected  subcutaneously  with  50  mgr. 
showed  no  lesion  on  death  three  years  after. 

Vitality  in  Culture. — The  bacillus  was  found  alive  in  cul- 
ture after  1067  days  (nearly  3  years). 

Chemical  Properties. — The  investigators  were  unable 
to  detect  any  definite  and  constant  bio-chemical  character 
by  which  tubercle  bacilli  of  one  type  can  be  differen- 
tiated from  those  of  nother. 

BOVINE  TUBERCULOSIS. — No  further  special  investi- 
gation has  been  taken  beyond  that  recorded  in  the  second 
Interim  Report,  where  in  30  cases  of  the  natural  disease  in 
bovines,  only  one  form  or  type  of  Bacillus  tuberculosis  was 
found. 

HUMAN  TUBERCULOSIS. — In  all,  128  cases  of  all 
forms  of  tuberculosis  in  man  were  investigated.  Twenty 
of  these  were  of  lupus,  and  are  treated  separately  because 
the  recognition  of  the  type  of  bacillus  was  surrounded 
with  special  difficulties.  The  108  cases  remaining  are 
tabulated  in  full  in  the  Report,  and  briefly  in  Table  I., 
page  294. 

Of  the  cases  of  primary  abdominal  tuberculosis,  the  ages 
at  death  were  as  shown  in  Table  II.,  p.  294.  Twenty-four 
of  the  deaths  were  from  some  form  of  tuberculosis,  the 
others  from  non-tuberculous  affections.  Of  15  of  these 
cases,  in  which  a  plurality  of  lesions  were  examined  on 
exactly  parallel  lines,  9  yielded  none  but  human  tubercle 
bacilli.  In  12  cases  in  which  a  single  lesion  in  each  instance 
was  examined  (mesenteric  glands  in  II,  cervical  gland  in  1), 
4  yielded  human  and  8  bovine  tubercle  bacilli.  Two  cases 
yielded  mixed  human  and  bovine  types,  in  one  from  the 
mesenteric  glands  alone,  and  in  the  other,  also  from  the 
retroperitoneal  glands. 

The  cervical  gland  cases  show  3  bovine  infections  out  of 


294         PUBLIC    HEALTH    BACTERIOLOGY 

Table  I.- — -Cases  of  Human  Tuberculosis  other  than  Lupus 


Type 

of  Bacillus 

Nature  of  Cases 

Cases 

found 

Bovine 

Human 

Mixed 

i.  Primary  pulmonary  tuberculosis: 

(phthisis  pulmonalis) 

A .  Tissues  examined  post  mortem 

r(lung   in    13    and   bronchial 

gland  alone  in  1) 

14 

— 

M 

— 

B.  Sputum  from  other  cases 

28 

2 

26 

— 

2.  General    tuberculosis :     various 

tissues 

3 



3 

— . 

3.  Tuberculous   meningitis :    cerebro- 

spinal fluid  in  2 

3 

■ 

3 

— 

4.  Bronchial  gland  tuberculosis 

5 



3 

2 

5.  Cervical  gland  tuberculosis  : 

(removed  by  operation) 

9 

3 

6 



6.  Primary  abdominal  tuberculosis  : 

mesenteric    glands    and    other 

tissues 

29 

M 

13 

2 

7.  Joint  and  bone  tuberculosis  : 

scrapings  and  abscesses 

14 

— 

13 

I 

8.  Tuberculosis  of  testicle  (1),  kidney 

(1),  and  suprarenal  (1)    . . 

3 

— ■ 

3 



Totals 

108 

19 

84 

5 

Notes. — The  cases  in  group  1  A  were  all  clinically  cases  of  con- 
sumption, in  which  death  resulted  from  the  pulmonary  disease. 

The  two  patients  who  had  bovine  tubercle  bacilli  in  their  sputum 
died  subsequently,  but  no  post-mortem  examination  was  obtained. 
The  ages  in  the  sputum  cases  were  :  16  to  25  years,  19  cases  ;  26 
to  33  years,  8  cases  ;   and  50  years,  1  case. 


Table  II. — Primary 

Abdominal  Tuberculosis. 

Type 

of  Virus  found 

PRESENT 

Bovine 

Human 

Mixed 

1  to  3  years 

3  to  5      „    ■ 

4  to  5      „    . 

7  »    • 

8  „     . 
15          „     • 
18          „     • 
70          „    • 

IO 

3 

I 

8 
3 

1 

1 

I 
I 

18 
3 

3 

Tc 

>tals 

M 

13 

2 

29 

NON-SPORING    BACILLI 


295 


9  investigated,  and  these  too  are  ascribed  to  pharyngeal 
or  buccal  infection,  that  is,  alimentary  infection.  With 
the  primary  abdominal  cases,  this  gives  38  cases  of 
tuberculosis  of  presumably  alimentary  infection,  in  which 
the  bovine  bacillus  alone  was  found  in  17  instances,  the 
human  in  19,  and  a  mixed  infection  in  2. 
Summary. 


Cases 
Exam- 
ined. 

Type  of  Virus  Found. 

Bovine. 

Human. 

Mixed. 

Avenue  of  infection  : 

(a)  Respiratory    tract     (presum- 
ably)                     

(b)  Alimentary    tract     (presum- 
ably)                     

47 

38 

2 
*7 

43 
19 

2 

2 

Age :                   

(a)  Adolescents  and  adults 

(6)   Children           

55 
53 

3 
16 

50 
34 

2 

3 

Lupus. — Out  of  20  cases  investigated,  the  virus  was 
decided  to  be  bovine  in  9  cases,  and  to  be  the  human 
type  in  11.  All  but  three  of  the  cases  presented  difficulty 
in  the  detection  of  the  type  of  bacilli.  One  was 
undoubtedly  bovine  by  the  already  decided-on  tests,  two 
were  likewise  human  ;  but  the  remaining  17  furnished 
bacilli  which  conformed  to  the  one  type  in  some  particulars 
and  to  the  other  in  other  particulars.  Thus  the  other 
8  finally  called  bovine  showed  the  cultural  characters  of 
that  type,  but  a  lowered  virulence  for  the  calf  and  also 
for  the  rabbit,  monkey,  and  guinea-pig.  Two  had  their 
virulence  raised  by  passage  through  the  calf  and  rabbit, 
bringing  it  up  to  that  of  the  bovine  type.  Those  ascribed 
to  the  human  type  (11),  but  not  typically  so,  had  lowered 
virulence  for  all  the  test  animals  or  some  of  them.  The 
general  result  therefore  seems  to  point  to  the  existence  of 
bacilli  outside  the  types  chosen  ;  these  may  be  new  types, 
or  members  of  the  other  types  with  degraded  virulence. 

Tuberculosis  in  Swine. — In  59  cases  investigated  of 


296         PUBLIC    HEALTH    BACTERIOLOGY 

tuberculosis  of  all  kinds  in  swine,  the  bovine  virus  was 
found  in  50  cases,  the  human  in  3,  the  avian  in  5,  and  a 
mixed  avian  and  bovine  virus  in  1  case. 

In  33  of  these  cases  the  tuberculosis  was  generalized, 
and  in  32  of  these  the  infection  was  bovine,  the  other 
being  the  one  of  mixed  bovine  and  avian  infection.  The 
other  26  cases  showed  local  tuberculosis. 

Conclusions  :  All  three  types  of  bacilli  are  capable  of 
infecting  the  pig,  but  the  bovine  bacillus  is  the  one  much 
most  frequently  found,  and  in  many  instances  it  produces 
a  severe  and  generalized  disease. 

Tuberculosis  in  Horses. — In  most  cases  of  tuberculosis 
in  horses  it  is  primarily  an  affection  of  the  glands  and 
organs  in  connection  with  the  alimentary  tract ;  the 
abdominal  organs  chiefly.  This  was  so  in  the  five  cases 
investigated.  In  all,  bacilli  were  recovered  which  were  of 
the  bovine  type  culturally ;  and  three  of  them  also  had 
the  bovine  virulence.  The  other  two  had  diminished 
virulence  for  the  test  animals  in  the  test  doses ;  passage 
experiments  raised  this  to  that  of  the  bovine  virus. 

Tuberculosis  in  other  Animals.  —  In  a  gnu  which 
died  from  generalized  tuberculosis  in  the  London  Zoological 
Gardens,  the  human  virus  only  was  found. 

In  an  antelope,  killed  when  ill,  in  the  same  Gardens, 
where  it  had  been  many  years  in  captivity,  extensive 
tuberculosis,  with  cavities  in  the  lungs,  was  found.  The 
human  virus  alone  was  isolated. 

In  a  rhesus  monkey,  killed  when  ill  at  the  quarantine 
station  at  Isleworth,  tuberculosis  of  the  lungs  was  found, 
and  some  elsewhere.  Here  too  only  the  human  virus 
could  be  got. 

A  chimpanzee  died  of  acute  miliary  tuberculosis  at  the 
quarantine  station.  The  tuberculosis  started  from  the 
alimentary  tract,  and  from  a  mesenteric  gland  the  human 
virus  was  obtained. 

A  cat,  suffering  from  naturally  acquired  tuberculosis, 
was  investigated.  A  mesenteric  gland  gave  a  bacillus  of 
the  bovine  type  in  growth  and  virulence. 

Tuberculosis  in  Birds.  —  In  9  cases  investigated 
(3  fowls,  3  pheasants,  1  pigeon,  1  demoiselle  crane,  1 
Senegal  touracou)   of   tuberculosis  occurring  naturally  in 


NON-SPORING    BACILLI  297 

birds,  in  every  one  the  virus  found  was  of  the  avian  type. 

In  tuberculosis  in  pigs,  the  avian  virus  was  found  on 
6  occasions  in  the  submaxillary  lymphatic  glands  ;  in 
5  alone,  and  in  I  case  in  association  with  the  bovine  virus. 

(Query. — Is  this  due  to  special  exposure  of  the  pig  to  the 
farmyard  dust,  and  to  its  high  body  temperature  ?) 

Behaviour  of  the  Bacilli  and  their  Fate  in  the 
Tissues  of  Inoculated  Animals. — From  a  number  of 
experiments  detailed,  the  Reporters  conclude  that  after 
subcutaneous  inoculation  of  human  and  bovine  viruses, 
rapid  and  abundant  distribution  of  bacilli  over  the  body 
takes  place,  provided  the  dose  is  large  and  the  tissue  condi- 
tions of  the  animal  such  as  to  allow  the  inoculation  to  take 
full  effect.  This  is  essentially  a  mechanical  dispersion  by 
the  blood  and  lymph  channels  of  a  considerable  proportion 
of  injected  bacilli,  and  occurs  speedily  after  their  insertion. 
The  resulting  acute  tuberculosis  is  at  first  in  strong  contrast 
to  the  similar  but  more  slowly  developing  generalized 
disease  induced  by  smaller  experimental  doses,  or  such  as 
occurs  most  commonly  in  nature.  This  more  slowly 
developing  form  is  due  to  a  dispersion  of  bacilli,  but  not 
necessarily  those  primarily  invading  the  animal ;  more  pro- 
bably their  progeny,  which  have  been  able  to  get  through 
the  barriers  and  into  the  blood-stream. 

Feeding  Experiments. — In  two  pigs  fed  with  large 
doses  of  bovine  virus,  the  bacilli  were  demonstrated  (by 
inoculation  of  guinea-pigs)  in  the  submaxillary  and  mes- 
enteric glands  and  lungs  in  seven  and  thirteen  days  after 
ingestion,  but  not  in  the  other  organs.  In  seven  pigs  fed 
with  human  virus,  in  from  two  to  twelve  days  after 
ingestion  the  same  distribution  was  proved  in  two ;  in 
the  other  five  the  virus  was  found  in  the  glands  alone. 
In  a  goat,  eight  days  after  ingestion  of  human  bacilli, 
they  were  found  in  the  submaxillary  glands,  mesenteric 
glands,  and  lung.  In  a  cat  fed  with  the  same  bacilli, 
in  nine  days  they  were  found  in  the  submaxillary 
and  mesenteric  glands,  lung,  liver,  and  spleen.  Three 
rhesus  monkeys,  each  fed  with  50  mgr.  of  human  tubercle 
bacilli,  were  killed  ;  1  in  two  days,  showed  no  bacilli  in 
the  glands  or  internal  organs;  1  in  four  days,  showed 
bacilli  in  the  mesenteric  glands,  liver,  spleen,  and  lungs 


298  PUBLIC     HEALTH     BACTERIOLOGY 

and  the  third  in  six  days,  showed  bacilli  in  the  sub- 
maxillary and  mesenteric  glands  and  spleen,  the  lung 
condition,  owing  to  the  premature  death  of  the  guinea- 
pigs  used  for  the  test,  not  being  determined. 

Excretion  of  tubercle  bacilli  in  milk  was  tested  by 
injection  of  cultures  into  healthy  cows  and  goats. 

Subcutaneous  injection  of  ioo  mgr.  of  bovine  culture  into 
a  healthy  milch  cow  caused  its  death  thirty  days  later  of 
general  tuberculosis.  The  udder  was  normal  both  to  the 
naked  eye  and  on  microscopical  examination,  yet  guinea- 
pigs  fed  with  the  animal's  milk,  by  the  end  of  the  first  week 
after  injection  and  subsequently,  developed  tuberculosis. 

Two  other  cows  were  injected  with  human  tubercle 
bacilli.  One  received  ioo  mgr.  subcutaneously  ;  tubercle 
bacilli  were  recovered  from  her  milk  in  twenty-four 
hours,  and  the  milk  in  small  doses  caused  tuberculosis  in 
guinea-pigs  at  every  time  of  testing  right  up  to  155  days 
later,  when  the  cow  was  killed  and  the  udder  was  found 
normal.  The  other  received  10  mgr.  intravenously,  and  the 
milk  contained  the  bacilli  in  twenty-four  hours  and  up  to 
fourteen  days  later,  but  not  subsequently.  The  animal 
was  killed  in  182  days,  and  showed  no  tuberculous  lesions. 

Six  milch  goats  were  similarly  tested  with  bovine  and 
human  bacilli,  with  confirmatory  results. 

In  12  experiments  on  heifers,  subcutaneous  injection  of 
human  virus  (2),  lupus  virus  (8),  and  bovine  virus  (2),  in 
50  to  100  mgr.  doses,  the  heifers  were  killed  in  62  to  127 
days  afterwards,  and  in  eight  cases  tubercle  bacilli  were 
found  in  the  sinuses  of  the  undeveloped  udder  of  the 
animals  ;  in  four,  in  such  numbers  as  to  suggest  multipli- 
cation in  these  sinuses. 

Modification  of  Bacilli. — Tests  by  cultural  processes 
and  by  long-continued  passage  through  animals  have 
failed  to  effect  any  change  of  type  from  bovine  to  human, 
bovine  to  avian,  human  to  avian,  and  vice  versa.  Certain 
difficulties  were  encountered  with  bacilli  in  lupus  and  in 
the  horse,  but  while  the  Reporters  think  that  further 
investigations  may  possibly  disclose  additional  variations 
in  the  types  of  bacilli,  they  do  not  as  a  result  of  their 
investigations  feel  disposed  to  add  a  plurality  of  new 
types  to  the  three  already  described  by  them.     They  are 


NON-SPORING    BACILLI  299 

not  prepared  to  deny  that  the  transmutation  of  one  type 
into  another  may  occur  in  nature,  in  view  of  the  instances 
in  which  one  and  the  same  human  body  yielded  both 
types. 

Replies  to  the  Terms  of  Reference. — The  questions 
referred  to  them  for  investigation  and  report  were  as 
follows  : — 

(i)  "  Whether  the  disease  in  animals  and  in  man  is  one  and 
the  same.  (2)  Whether  animals  and  man  can  be  recipro- 
cally infected  with  it.  (3)- Under  what  conditions,  if  at 
all,  the  transmission  of  the  disease  from  animals  to  man 
takes  place,  and  what  are  the  circumstances  favourable 
or  unfavourable  to  such  transmission." 

1.  Morphologically,  as  grown  on  serum,  the  human  and 
bovine  types  described  are  indistinguishable,  but  they  are 
appreciably  different  in  respect  of  their  cultural  characters 
and  their  capacity  for  causing  disease  in  various  species 
of  animals.  The  question  of  the  identity  or  non-identity 
of  these  two  types  clearly  depends  therefore  upon  the 
importance  which  it  is  permissible  to  attach  to  their 
cultural  and  pathogenic  differences,  and  this  depends  on 
the  fixity  or  variability  of  the  differences  in  question. 
Though  in  the  investigations  no  case  was  observed  in  which 
the  mode  of  growth  of  one  type  was  so  modified  that 
it  was  indistinguishable  from  the  mode  of  growth  of  the 
other  type,  yet  the  bacilli  referred  to  as  the  bovine  type 
show  so  much  variety  among  themselves  as  to  luxuriance 
of  growth,  that  the  gap  which  separates  those  of  that  group 
which  grow  most  luxuriantly  from  the  human  type,  is 
not  a  wide  one. 

Again,  as  regards  pathogenicity,  it  is  more  a  matter  of 
degree  than  of  difference.  The  bovine  tubercle  bacillus 
produces  a  fatal  tuberculosis  in  cattle,  rabbits,  guinea- 
pigs,  chimpanzees,  monkeys,  goats,  and  pigs.  The  human 
tubercle  bacillus  readily  produces  a  fatal  tuberculosis  in 
guinea-pigs,  chimpanzees,  and  monkeys,  and  in  large 
closes,  only  slight  and  non-progressive  lesions  in  cattle, 
goats,  and  pigs.  Its  effects  on  rabbits  are  not  uniform, 
for  while  in  the  majority  of  cases  these  animals  are 
only  slightly  affected,  in  some  cases  extensive  and  fatal 
tuberculosis  results. 


300         PUBLIC    HEALTH    BACTERIOLOGY 

In  other  words,  guinea-pigs,  chimpanzees,  and  monkeys 
are  all  highly  susceptible  to  the  effects  of  human  or  bovine 
tubercle  bacillus ;  and  the  diseases  produced  in  these 
animals  by  both  types  are  histologically  and  anatomically 
identical. 

In  man,  experiment  is  not  permissible,  and  no  oppor- 
tunity has  offered  of  generalized  disease  set  up  by  accidental 
infection  with  the  bovine  tubercle  bacillus.  Nevertheless 
many  cases  of  fatal  tuberculosis  caused  by  the  bovine 
tubercle  bacillus  and  nothing  else  have  been  investigated. 
Compared  with  parallel  cases  caused  by  the  human  tubercle 
bacillus,  the  two  groups  of  cases  were  alike  in  their  clinical 
histories  and  their  fatal  termination,  and  were  indis- 
tinguishable anatomically  when  the  lesions  were  examined 
after  death.  Man  must  therefore  be  added  to  the  list  of 
animals  notably  susceptible  to  bovine  tubercle  bacilli. 

Are  these  two  types,  then,  varieties  of  the  same  organism  ? 
This  is  the  conclusion,  in  spite  of  the  failure  to  transmute 
the  one  into  the  other.  And,  as  a  corollary,  the  lesions 
they  produce,  whether  in  man  or  in  other  mammals,  are 
manifestations  of  the  same  disease.  Whatever  difference 
of  opinion  may  be  held  on  this  conclusion,  in  a  considerable 
proportion  of  cases  of  human  tuberculosis  the  disease  is 
one  and  the  same  as  bovine  tuberculosis,  being  caused  by 
bacilli  which  are  in  every  respect  indistinguishable  from 
the  bacilli  which  are  the  cause  of  tuberculosis  in  cattle. 
In  all  such  cases  therefore  the  disease  must  unquestionably 
be  pronounced  as  one  and  the  same. 

As  regards  avian  tuberculosis,  there  does  not  appear  to 
be  sufficient  evidence  at  present  to  answer  the  question  in 
the  affirmative. 

2.  The  conclusion  reached  is  that,  excluding  the  fowl 
and  other  birds,  mammals  and  man  can  be  reciprocally 
infected  with  tuberculosis.  The  transmission  to  man  has 
been  conclusively  shown  by  the  study  of  fatal  cases  of 
tuberculosis,  mostly  in  children ;  and  from  man  to 
mammals,  by  feeding  experiments. 

3.  Conclusions  : — 

(i.)  Unmodified  avian  tubercle  bacillus  is  a  negligible 
factor  in  the  production  of  human  tuberculosis. 

(ii.)  It  cannot  be  affirmed  with  confidence  that  man  is 


NON-SPORING    BACILLI  301 

wholly  free  from  risk  of  infection,  through  animal  food, 
with  that  type  of  bacillus  to  which  he  is  most  prone, 
namely  the  human  type ;  though  the  degree  of  danger  to 
him  in  this  sense  must  for  the  present  remain  undetermined. 
The  pig,  though  experimentally  it  fostered  human  bacilli 
in  a  minor  degree  only,  may  have  to  be  regarded  as  a 
possible  source  of  this  kind  of  infection,  since  particular 
glands  of  the  pig's  body,  in  which  human  tubercle  bacilli 
have  occasionally  been  found,  are  likely  to  enter  into 
certain  prepared  foods. 

(iii.)  (a)  The  pig  is  a  potential  source  of  infection  of  man 
with  bovine  tubercle  bacilli.  This  bacillus  was  present  in 
50  -f- 1  cases  of  tuberculosis  in  pigs,  out  of  59  cases  of 
tuberculosis  investigated.  In  32  +  1  of  these  cases,  it 
caused  generalized  tuberculosis,  and  in  18  cases,  local 
tuberculosis.  (The  -f  1  case  was  one  of  mixed  bovine  and 
avian  infection.)  There  is  no  reason  to  suppose  that  the 
bovine  tubercle  bacilli  are  rendered  less  infective  to  human 
beings  by  previous  residence  in  the  tissues  of  the  pig. 

(b)  The  actual  number  of  cases  representing  the  various 
clinical  manifestations  of  tuberculosis  commonly  found  in 
man,  on  which  the  conclusions  are  based,  is.  128.  So  far 
as  these  have  been  examples  of  tuberculosis  in  adults, 
and  especially  when  they  have  been  cases  of  pulmonary 
tuberculosis,  the  lesions  of  the  disease,  when  fatal,  have 
been  referable  to  the  human  tubercle  bacillus,  with  but 
few  exceptions. 

In  human  abdominal  tuberculosis,  the  experience  has 
been  very  different,  especially  as  regards  children.  Of 
young  children,  dying  of  primary  abdominal  tuberculosis, 
the  fatal  lesions  could  be  referred  to  the  bovine  tubercle 
bacillus,  and  it  alone,  in  nearly  one-half  of  the  cases. 

In  cervical-gland  tuberculosis,  in  children,  and  often 
also  in  adolescents,  a  large  proportion  of  the  cases  examined 
could  be  referred  to  the  bovine  tubercle  bacillus. 

In  lupus,  too,  in  the  cases  examined  occurring  in 
adolescents  and  children,  the  amount  of  infection  with 
the  bovine  type  was  marked. 

Whatever  therefore  may  be  the  animal  source  of 
infection  with  the  bovine  type  of  bacillus  in  adult  and 
adolescent  mankind,  there  can  be  no  doubt  that  a  consider- 


302         PUBLIC    HEALTH    BACTERIOLOGY 

able  proportion  of  the  tuberculosis  affecting  children  is 
of  bovine  origin,  more  particularly  that  which  affects 
primarily  the  abdominal  organs  and  the  cervical  glands, 
and  further  that  primary  abdominal  tuberculosis,  as  well 
as  tuberculosis  of  the  cervical  glands,  is  commonly  due  to 
ingestion  of  tuberculous  infective  material. 

In  what  way  are  children  most  likely  to  obtain  a  large 
and  fatally  infective  dose  of  tubercle  bacilli  ?  To  this 
question  there  can  be  but  one  answer,  namely,  the  evidence 
accumulated  goes  to  demonstrate  that  a  considerable 
amount  of  tuberculosis  in  children  is  to  be  ascribed  to 
infection  with  bacilli  of  the  bovine  type,  transmitted  in 
meals  consisting  largely  of  the  milk  of  the  cow. 

The  child  may  be  subjected  to  this  feeding  with  infective 
material,  and  not  develop  a  fatal  tuberculosis  ;  but  still  be 
injured,  although  it  recover.  Many  cases  of  abdominal 
tuberculosis  in  children  recover,  and  the  proportion  of 
bovine  to  human  bacilli  in  these  has  not  been  estimated  ; 
and  of  cervical-gland  tuberculosis,  nearly  all  make  some 
kind  of  recovery,  with  varying  degrees  of  disfigurement ; 
and  a  similar  statement  may  be  applied  to  lupus. 

In  adult  and  adolescent  mankind  (excluding  lupus,  in 
which,  out  of  10  cases,  three  yielded  bacilli  culturally 
bovine  but  with  less  virulence  for  the  calf  and  rabbit  than 
the  bovine  tubercle  bacillus),  fatal  lesions  due  to  bovine 
bacilli  have  been  found,  rarely  in  adolescents,  and  extremely 
rarely  in  adults.  Yet,  although  of  55  cases  scrutinized  of 
tuberculosis  in  adults  and  adolescents,  only  5  yielded 
bovine  bacilli,  it  cannot  be  said  that  this  figure  adequately 
represents  the  proportion  of  like  cases  among  the  tubercu- 
lous population  generally. 

In  view  of  the  evidence  adduced,  the  following  pronounce- 
ments on  administrative  measures  required  at  present  to 
obtain  security  against  transmission  of  bovine  tubercle 
bacilli  by  means  of  food,  are  called  for  : — 

In  the  interests  of  infants  and  children,  and  for  the  reasonable 
safeguarding  of  the  public  health  generally,  it  is  urged  that 
existing  regulations  and  supervision  of  milk  production  and 
meat  preparation  be  not  relaxed. 

On   the  contrary,   Government   should    cause    to    be    enforced 


NON-SPORING    BACILLI  303 

throughout  the  kingdom  food  regulations,  planned  to  afford 
better  security  against  the  infection  of  human  beings  through 
the  medium  of  articles  of  diet  derived  from  tuberculous  animals. 
More  particularly  it  is  urged  that  action  in  this  sense  should 
be  taken,  in  order  to  avert  or  minimize  the  present  danger 
arising  from  the  consumption  of  infected  milk. 

Certain  facts  observed  in  reference  to  the  elimination  of 
bovine  tubercle  bacilli  by  the  cow  in  her  milk,  are  of  such 
importance  that  they  formed  the  subject  of  the  third 
Interim  Report,  and  deserve  repetition  here. 

i.  Bovine  tubercle  bacilli  are  apt  to  be  abundantly 
present  in  milk,  as  sold  to  the  public,  when  there  is 
tuberculous  disease  of  the  udder  of  the  cow  from  which 
it  has  been  obtained.  This  fact  is  generally  recognized, 
though  not  adequately  guarded  against. 

2.  Bovine  tubercle  bacilli  may  also  be  present  in  the 
milk  of  tuberculous  cows  presenting  no  evidence  whatever 
of  disease  of  the  udder,  even  when  examined  post  mortem. 

3.  In  tuberculous  cows,  the  milk  leaving  the  udder  may 
not  contain  tubercle  bacilli,  and  yet  it  may  and  frequently 
does  become  infective  by  contamination  with  the  faeces  or 
uterine  discharges  of  such  diseased  animal. 

Convinced  that  measures  for  securing  the  prevention  of  the 
ingestion  of  living  bovine  tubercle  bacilli  with  milk  would  greatly 
reduce  the  number  of  cases  of  abdominal  and  cervical  gland 
tuberculosis  in  children,  the  Reporters  advise  that  such  measures 
should  include  the  exclusion  from  the  food  supply  of  the  milk 
of  the  recognizably  tuberculous  cow,  irrespective  of  the  site  of 
the  disease,  whether  in  the  udder  or  in  the  internal  organs. 

(A  memorandum  is  appended  in  which  reference  is 
made  to  immunity  experiments,  on  which  no  opinion  is 
expressed,  but  which  are  fully  reported  in  Vol.  iii.  of 
Appendix  to  this  Report ;  and  to  several  other  subsidiary 
experiments.) 


CHAPTER  XIV. 
SPORING     BACILLI. 

Sporing  bacilli  are  comprised  in  two  groups  : — 

i.  Aerobic  (facultative  anaerobes).  Non-motile  :  anthrax, 
anthracoides  and  radicosus.  Sluggishly  motile  :  mycoides, 
ramosus,  vulgatus,  mesentericus.  Actively  motile  :  subtilis, 
megatherium. 

AH  the  above  are  Gram-positive ;  gelatin-liquefying ; 
non-indol-forming,  and  non-gas-forming  in  glucose  or 
lactose ;  coagulate  milk  slowly  with  little  acid  and  then 
digest  the  clot ;  digest  blood  serum. 

2.  Anaerobic  (strictly). — Subcutaneous  injection  into 
animals  causes  : — 

(i).  No  particular  symptoms  at  site  of  inoculation,  but 
absorption  of  the  soluble  toxin  causing — (a)  general 
symptoms  of  tetanus,  B.  tetani ;  (b)  botulism,  pupillary 
symptoms,  paralysis  of  tongue  and  pharynx,  cardiac  and 
respiratory  failure,  B.  botulinus. 

(ii).  Local  symptoms  marked  at  the  site  of  inoculation, 
causing  haemorrhagic  emphysematous  oedema  ;  (a)  motile  ; 
spores  oval  and  central,  B.  cedematis  maligni ;  spores  oval 
and  excentric,  B.  anthracis  symptomatici ;  spore  near  one 
end,  B.  enteritidis  sporogenes ;  (b)  non-motile;  B.  aerogenes 
capsulatus  of  Welch  and  Nuttall. 

SPORE-BEARING    AEROBIC     BACILLI. 

Bacillus  Anthracis  is  the  cause  of  anthrax,  a  disease 
primarily  of  the  herbivora,  cattle  and  sheep,  but  occurring 
also  in  horses,  pigs,  and  goats.  Man  is  susceptible,  and 
contracts  it  either  directly  from  the  living  or  dead  animal, 
or  from  hides,  wool,  horse-hair,  or  dust  arising  from  these. 
It  assumes  two  forms,  external  anthrax  or  malignant 
pustule,  and  internal  anthrax  which  in  man  takes  the 
form  of  wool-sorter's  disease  and  the  form  of  intestinal 
anthrax,  in  which  the  symptoms  are  more  like  those  of 
acute  poisoning. 

In  human  anthrax,  bacterial  invasion  of  the  blood  only 


SPORING    BACILLI  305 

occurs  late  in  the  disease  ;  in  animals,  on  the  contrary,  the 
blood-invasion  is  early. 

The  Algerian  sheep  and  the  white  rat  have  a  high  degree 
of  immunity.  Pollender  first  described  the  anthrax 
bacillus  as  occurring  in  the  blood  of  animals  succumbing 
to  splenic  fever.  Rayer  and  Davaine  repeated  the 
observation  in  1850  (a  year  later) ;  Brauell,  in  1857,  found 
the  bacilli  in  the  blood  of  a  man  affected  with  anthrax, 
and  Davaine  gave  everything  but  absolute  proof  that  they 
were  the  exciting  cause  of  anthrax.  Koch,  by  succeeding 
in  getting  a  pure  culture  on  the  aqueous  humour  of  an  ox's 
eye,  was  able  to  prove  its  specificity.  He  also  added 
largely  to  the  knowledge  of  its  life-history,  and  particularly 
to  the  mode  of  formation  of  spores. 

Description. — B.  anthracis  is  a  straight  rod,  non-motile, 
with  square  or  concave  ends,  4-5  to  10  micra  long  by  1  to 
1-5  micron  thick;  forming  chains  in  cultures,  and  sporing 
by  oval  spores  one  to  each  rod ;  placed  about  the  centre  of 
the  bacillus,  and  of  about  the  same  diameter,  and  highly 
retractile.  Gram-positive ;  gelatin-liquefying,  and  said  at 
times  to  possess  a  capsule  when  recovered  from  tissues  or 
blood,  or  grown  on  latter. 

Cultures. — Grows  well  on  all  media,  best  at  37-5°  C, 
but  also  from  12 °  to  45 °  C. 

In  broth:  a  heavy  flocculent  sediment,  slight  pellicle, 
remainder  clear. 

In  gelatin  stab :  an  invreted  fir-tree  growth,  with  gradual 
fluidification. 

In  gelatin  plate :  colonies  develop  within  24  to  48  hours 
as  opaque  white  pin-head  discs,  later  becoming  larger  and 
less  regular,  and  under  the  microscope  showing  a  hair-like 
tangle  of  threads — the  so-called  Medusa  head. 

In  agar  plate :  the  colonies  magnified  thirty  times  show 
wavy  wreaths  like  locks  of  hair,  the  whole  colony  being 
probably  one  long  thread.  Such  colonies  are  very  suit- 
able for  making  impression  preparations,  and  in  such  the 
wreaths  are  seen  to  be  made  up  of  bundles  of  long  filaments 
lying  parallel  with  one  another,  each  filament  consisting  of 
a  chain  of  bacilli. 

On  potato  :  a  thick  white  felted  mass,  useful  for  studying 
porulation. 

20 


306         PUBLIC    HEALTH    BACTERIOLOGY 

Spores. — Only  produced  in  the  presence  of  oxygen 
(free),  and  hence  not  formed  in  blood  of  infected  animals 
while  in  the  unopened  vessels  or  tissues.  For  this  reason 
it  is  advised  to  cut  into  an  animal  dead  of  anthrax  as 
little  as  possible,  and  to  be  specially  careful  not  to  spill 
the  blood.  The  spores  are  very  resistant,  keeping  for 
twenty  years.  They  are  killed  by  dry  heat  at  1400  C. 
(2480  F.)  in  3  hours,  and  live  steam  at  ioo°  C.  in  5  to  10 
minutes,  or  boiling  water  for  1 J  hours.  Their  behaviour  to 
chemical  disinfectants  is  variable,  some  strains  resisting 
1-20  carbolic  acid  for  forty  days,  while  others  are  destroyed 
by  the  same  solution  in  two  days.  Corrosive  sublimate, 
1-2000,  kills  most  strains  in  40  minutes.  Direct  sunlight 
destroys  anthrax  spores  within  6  to  12  hours.  Creolin  (10 
per  cent)  kills  anthrax  bacilli  in  10  to  20  minutes,  but 
anthrax  spores  can  survive  in  a  60  per  cent  solution  of 
creohn.  Freezing  has  little  effect  on  their  vitality.  Spores 
are  formed  best  at  300  C,  and  by  keeping  the  bacilli  at 
420  C.  for  eight  days,  the  power  of  sporulation  is  lost,  and 
is  only  regained  by  passing  the  bacilli  through  a  series  of 
animals. 

Anthrax  spores  are  often  used  for  testing  the  value  of 
"  germicides."  To  do  this,  sterile  silk  threads  are  steeped 
in  an  emulsion  of  an  anthrax  culture  and  are  dried  over 
strong  sulphuric  acid  in  a  desiccator.  They  are  then 
placed  in  a  solution  of  the  "  germicide  "  for  a  certain  time, 
well  washed  with  water,  and  laid  on  the  surface  of  agar 
medium  or  dropped  into  broth,  and  incubated  to  see  if  any 
growth  occurs.  The  culture  used  is  first  tested  for  spore 
formation. 

Pathogenicity. — For  man  :  great.  For  animals  :  mainly 
for  cattle  and  sheep.  In  the  German  Empire  in  1899,  the 
following  cases  were  reported  :  3678  cattle,  307  sheep, 
282  horses,  61  swine,  and  6  goats. 

In  Great  Britain,  in  the  ten  years  1896  to  1905,  the 
total  reported  "  outbreaks  "  in  animals  were  6203,  and  the 
number  is  increasing.  In  man,  512  cases  were  reported  in 
1901  to  1910,  and  of  these  120  were  fatal.  Internal  anthrax 
is  usually  fatal.  In  the  external  form,  head  and  neck  cases 
show  a  mortality  of  85  per  cent  ;  and  hand  and  arm  cases 
12  per  cent. 


SPORING    BACILLI  307 

Rabbits,  guinea-pigs,  and  white  mice  are  all  very 
susceptible,  the  mice  most  so.  Rats  resistant,  especially 
the  white  rat ;  dogs  more  so.  Birds  are  highly  immune, 
also  amphibians  (but  toads  are  said  to  be  very  susceptible). 

Toxins. — No  toxins  have  yet  been  isolated,  though  it  is 
highly  probable  that  both  extra-  and  intra-cellular  toxins 
exist. 

Vaccination. — In  France,  a  death-rate  from  anthrax  of 
10  per  cent  among  sheep  and  5  per  cent  among  cattle 
compelled  attention  to  the  problem  of  providing  protection. 
Pasteur,  in  1881,  introduced  his  method  by  the  use  of  two 
vaccines  :  (1)  A  broth  culture  of  bacilli,  whose  virulence 
was  reduced  by  being  incubated  at  420  C.  for  twenty-four 
days,  and  so  made  non-fatal  to  guinea-pigs  but  still  fatal 
to  white  mice — premier  vaccin  ;  (2)  A  broth  culture,  incu- 
bated as  above  for  twelve  days,  which  would  kill  guinea- 
pigs  but  not  rabbits — deuxieme  vaccin. 

A  sheep  was  inoculated  in  the  subcutaneous  tissues  on 
the  inner  side  of  the  thigh  with  5  drops  of  the  premier 
vaccin.  Twelve  days  later  a  similar  inoculation  of  the 
deuxieme  vaccin  was  given,  and  fourteen  days  later  still 
an  injection  of  an  ordinary  virulent  culture  produced  no 
ill  result.  The  method  has  given  excellent  results,  and 
the  immunity  lasts  about  a  year. 

Passive  Immunization. — Sclavo  produced  a  serum  from 
highly  immunized  asses,  which  has  strongly  protective 
and  curative  properties,  and  is  used  in  the  treatment  of 
anthrax  in  man.  In  malignant  pustule,  four  doses  of  10  c.c. 
are  injected  into  the  abdominal  wall,  and  if  necessary 
repeated  on  the  following  day.  Sclavo  does  not  advise 
excision  of  the  pustule.  Sobernheim  uses  serum  from 
sheep. 

Isolation  of  B.  anthracis  from  hairs,  etc.  :  Add  5  grm. 
to  broth  and  shake.  Incubate  :  not  a  pure  culture.  Heat 
to  8o°  C.  for  30  minutes  ;  all  non-sporing  organisms  killed. 
Take  twenty  samples  of  1  c.c.  on  agar  and  grow.  Infect 
animals  and  see  if  pathogenic.     Plate  again  on  second  day. 

Diagnosis.  1.  In  a  case  suspected  to  be  malignant 
pustule,  diagnose  by  (1)  Making  films  from  the  fluid  in  the 
vesicles  or  from  scrapings,  and  staining  with  watery  methy- 
lene-blue,  and  also  by  Gram  (be  careful  in  scraping  a  pustule 


308         PUBLIC    HEALTH    BACTERIOLOGY 

before  excision,  not  to  manipulate  it  roughly,  or  bacteria 
may  enter  the  circulation) ;  (2)  Making  cultures  from 
similar  material,  by  successive  strokes  on  agar  tubes  or 
plates ;  (3)  Inoculation  of  the  cultures  into  a  guinea-pig 
or  mouse,  subcutaneously.  If  anthrax  bacilli  are  present 
the  animal  usually  dies  within  two  days,  and  post 
mortem  the  tissues  around  the  site  of  inoculation  show 
intense  inflammatory  oedema,  swelling,  and  gelatinous 
change,  with  small  haemorrhages.  On  microscopic  exami- 
nation, numerous  bacilli  are  seen.  The  internal  organs 
show  congestion  and  cloudy  swelling,  and  sometimes 
small  haemorrhages,  and  their  capillaries  contain  enor- 
mous numbers  of  bacilli,  so  that  they  appear  as  if 
injected  with  them.  The  spleen  is  notably  enlarged 
(especially  in  the  ox  dead  of  anthrax,  being  two  to  three 
times  its  natural  size,  hence  the  name  "splenic  fever"), 
is  of  a  dark-red  colour,  and  on  section  is  soft  and  friable, 
at  times  almost  diffluent.  Films  from  the  pulp  contain 
enormous  numbers  of  bacilli  mixed  with  red  cells  and 
leucocytes  of  the  lymphocyte  and  large  mononuclear 
varieties.  The  lymphatic  system  is  generally  much 
affected,  the  glands  and  vessels  being  swollen  and  containing 
bacilli  in  very  great  numbers.  The  intestines  are 
enormously  congested,  the  epithelium  is  more  or  less 
desquamated,  and  the  lumen  filled  with  a  bloody  fluid. 
(Muir  and  Ritchie.) 

2.  Methylene-blue  Reaction. — Depends  on  the  disintegra- 
tion of  the  capsules  of  the  bacilli,  which  occurs  when  these 
are  imperfectly  fixed.  It  serves  for  the  easy  recognition 
of  anthrax  bacilli  in  blood  and  other  bodily  fluids,  where 
putrefactive  and  other  bacilli  are  present.  Dry  a  loopful 
of  blood  on  a  slide  ;  hold  it  for  one  second  in  the  flame  ; 
repeat  three  times.  Stain  for  a  few  seconds  in  old  solution 
of  methylene-blue,  wash  in  water,  and  dry.  Examine  dry 
and  without  a  cover-glass,  when  between  and  near  the 
bacteria,  violet  or  reddish-purple  tinted  granular  or 
amorphous  matter  is  seen.     (M'Fadyean's  test.) 

Capsules  can  be  demonstrated  in  smear  preparations 
from  organs,  by  staining  in  2  per  cent  watery  solution  of 
methylene- violet  (heating).  Wash  in  water  for  2  seconds. 
Wash  in  1  per  cent  acetic  for  6  to  10  seconds.     Wash  in 


SPORING    BACILLI  309 

water  and  examine  in  water-drop.  Another  method  is  to 
stain  (without  fixing  film)  in  a  cold  saturated  solution  of 
gentian-violet  in  formalin.     Examine  in  water-drop. 

Prevention  of  Anthrax. — The  Home  Office  Order  No.  1293, 
dated  Dec.  12th,  1905,  on  this  subject,  is  made  under 
Section  79  of  the  Factory  and  Workshop  Act,  1901.  It 
provides  for  the  prevention  of  dust  from  wool  or  hair  by 
ordering  the  opening  and  sorting  to  be  done  only  (1)  after 
steeping  in  water  ;  or  (2)  over  an  efficient  opening  screen, 
with  mechanical  exhaust  draught,  in  a  room  set  apart  for 
the  purpose  and  in  which  no  other  work  than  opening  is 
carried  on.  Mohair,  other  than  Van  mohair,  only  needs 
to  be  sorted  over  a  down  draught.  Van  mohair,  Persian 
locks,  and  Persian,  must  all  be  steeped  before  being 
opened.  Alpaca,  pelitan,  East  Indian  cashmere,  Russian 
camelhair,  Pekin  camelhair  and  Persian  [or  so-called  Per- 
sian, if  to  be  sorted  or  willowed]  must  be  steeped  before 
opening,  or  opened  over  efficient  opening  screen.  It  also 
provides  for  the  use  of  overalls  and  respirators,  cubic 
space  per  person  (1000  c.  ft.),  temperature  of  room  (not 
lower  than  500  F.).  Treatment  of  cuts  and  sores,  washing 
of  hands,  burning  of  dust  and  other  refuse,  rules  for  treat- 
ment of  damaged  hair,  proper  kind  of  screen  and  board, 
lime-washing  of  rooms  yearly,  daily  disinfection  of  floors 
and  sweeping  thereafter,  etc.,  etc. 

Anthrax  orders  are  issued  by  the  Board  of  Agriculture 
under  the  Diseases  of  Animals  Act,  1894,  and  provide  for 
the  precautions  to  be  used  in  treatment  of  a  sick  animal, 
and  (1)  the  burial  at  a  depth  of  not  less  than  6  ft.,  with  1  ft. 
of  lime  beneath  and  above  the  carcase  of  an  animal  dead 
of  anthrax,  or  (2)  the  destruction  of  a  dead  animal  by 
heat  or  chemical  agents.  The  local  authority  must  carry 
out  the  provisions  of  the  order  in  these  and  other 
particulars. 

Bacteria  closely  resembling  B.  Anthracis  : — 

1.  B.  Anthracoides.  A  Gram-positive  bacillus;  ends 
more  rounded  than  those  of  B.  anthracis ;  growth  more 
rapid ;  gelatin  liquefaction  more  rapid ;  non-pathogenic. 
Otherwise  indistinguishable  from  B.  anthracis. 

2.  B.    Radicosus.     Cultivated     from     water-supplies. 


310         PUBLIC    HEALTH    BACTERIOLOGY 

Larger,  and  more  variation  in  size  of  individuals ;  grows 
best  at  room  temperature ;  non-pathogenic. 

3-  B.  Subtilis.  The  common  bacillus  of  hay  infusion, 
and  found  as  a  saprophyte  in  old  wounds  and  infected 
sinuses  ;  gives  a  heavy  tenacious  pellicle  in  broth  ;  spores 
germinate  equatorially  ;  gelatin  and  casein  are  liquefied 
more  rapidly  than  by  B.  anthracis  ;  is  actively  motile  in 
young  cultures  ;  non-pathogenic.  Pathogenicity  for  man 
is  now  alleged,  it  having  been  isolated  from  a  case  of 
panophthalmitis  in  pure  culture. 

4.  B.  Mycoides  (earth  bacillus).  Is  obtained  from  the 
surface  of  the  earth  of  cultivated  fields  or  gardens.  Grows 
best  at  180  C.,  and  on  gelatin  shows  a  mould-like  growth. 
About  the  size  of  B.  anthracis,  which  it  resembles  greatly. 
Grows  in  threads  ;  motile ;  non-pathogenic. 

5-  B.  Megatherium.  10  micra  x  2-5  micra.  First 
found  on  boiled  cabbage  leaves. 

6.  B.  Vulgatus  (potato  bacillus).  Grows  rapidly  on 
potatoes,  showing  marked  wrinkling.  Small  thick  rods 
with  rounded  ends,  in  pairs  or  fours. 

SPORE-BEARING     ANAEROBIC     BACILLI. 

Methods  of  Anaerobic  Culture. 

1.  Liquid  media  :  Boil  vigorously  for  15  minutes,  to 
drive  out  dissolved  oxygen  ;  cool,  and  put  layer  of  sterile 
oil  on  surface.     (Pasteur.) 

2.  Piece  of  sterile  mica  on  surface  of  agar  or  gelatin 
plates.     (Koch.) 

3.  Deep  inoculation  in  solid  media,  recently  boiled  for 
15  minutes  and  cooled  rapidly  in  ice,  to  prevent  absorp- 
tion of  oxygen.  It  is  advantageous  to  have  1  per  cent 
of  glucose  in  medium.  The  tubes  are  inoculated  by  deep 
stabs,  and  the  top  of  the  medium  is  covered  with  a  thin 
layer  of  agar,  gelatin,  or  oil.  and  the  tube  capped  with 
rubber  or  sealed  with  sealing-wax.  (Liborius.) 

4.  The  solid  medium  may  be  inoculated  before  it 
solidifies, — a  "  shake  culture  " — as  in  the  original  method 
of  Liborius.  The  colonies  which  develop  are  fished  out 
after  breaking  the  tube. 

5.  Tube   fitted   with    two-holed   cork   and   two   tubes. 


SPORING    BACILLI  311 

Pass  in  H  or  N  until  all  the  air  is  displaced,  and  then  seal 
ends  of  tubes  in  flame. 

6.  Buchner's  Tube. — A  wide  tube  with  a  constriction 
near  the  closed  end,  so  that  an  ordinary  culture  tube  can 
be  inserted  and  is  held  up  by  the  constriction.  In  use, 
i  grm.  of  solid  pyrogallic  acid  and  20  c.c.  of  10  per  cent 
KOH  are  put  into  the  bulb,  the  previously  inoculated  tube 
is  inserted  loosely  plugged,  and  the  Buchner  tube  is 
plugged  and  sealed  with  melted  paraffin  or  closed  with 
a  rubber  cap.  The  solution  in  bulb  absorbs  oxygen.  The 
method  can  be  used  without  the  special  tube,  with  a  tall 
glass  jar  or  a  desiccator.  It  is  better  to  add  the  alkali 
by  pipette  after  the  inoculated  tube  has  been  inserted. 

7.  Bulloch's  Method. — A  bell  jar  with  an  inlet  and  an  exit 
tube,  both  having  stopcocks.  The  bell  jar  is  firmly  bound 
down  to  a  glass  plate  by  ung.  resinae.  Before  fixing, 
a  glass  dish  is  set  on  the  plate,  and  3  to  4  grm.  of  pyro- 
gallic acid  are  heaped  to  one  side  of  it.  Culture  plates  or 
tubes  in  a  beaker  are  rested  on  a  tripod  stand  placed  in  the 
dish.  The  bell  jar  is  now  fixed  on  so  that  the  inlet  tube 
will  end  in  the  dish  at  the  side  away  from  the  pyrogallol. 
Hydrogen  is  passed  in,  and  then  a  solution  of  KOH 
(109  grm.  in  145  c.c). 

8.  Vacuum  Method. — Desiccator  with  stopcock.  Exhaust 
air  by  burning  alcohol  on  soaked  filter-paper  put  into  jar. 
Stopcock  is  needed  to  release  pressure  when  opening. 

B.  Tetani. 

The  cause  of  tetanus  or  lockjaw,  a  disease  characterized 
by  the  gradual  onset  of  general  stiffness  and  spasms  of  the 
voluntary  muscles,  beginning  in  the  jaw  muscles  and  those 
of  the  back  of  the  neck.  The  disease  is  usually  associated 
with  a  wound  received  four  to  fourteen  days  previously  and 
infected  with  earth  or  dung.  The  majority  of  cases  are  fatal. 

Kitasato  first  isolated  the  bacillus  (in  1889)  and  in 
this  fashion  : — Pus  from  the  local  suppuration  of  mice 
inoculated  from  a  human  case,  was  smeared  upon  the 
surface  of  agar  slants.  These  were  permitted  to  develop 
at  370  C.  for  24  to  48  hours.  At  the  end  of  this  time  the 
cultures  were  subjected  to  a  temperature  of  8o°  C.  for 
1  hour.     This  destroyed  all  non-sporulating  bacteria  as 


312         PUBLIC    HEALTH    BACTERIOLOGY 

well  as  aerobic  spore-bearers  which  had  developed  into  the 
vegetative  form.  Agar  plates  were  then  inoculated  from 
the  slants  and  incubated  in  a  hydrogen  atmosphere,  and 
on  these  tetanus  bacilli  grew.  Rosenbach  had  previously 
pointfd  out  the  terminal  spore  formation  of  B.  tetani, 
and  Nicolaier  had  described  the  bacillus,  but  could  not 
grow  it  in  pure  culture. 

Description. —  A  slender  bacillus,  2  to  5  micra  long  x 
0-3  to  0-8  micron  thick,  with  somewhat  rounded  ends.  In 
young  cultures  it  is  slightly  motile,  and  by  special  staining 
numerous  peritrichal  flagella  are  seen.  In  24  to  48  hours 
the  bacilli  develop  spores  which  are  at  full  size  three  to 
four  times  the  thickness  of  the  bacillus  in  diameter,  and 
are  formed  at  one  end  of  the  bacillus.  The  bacillus  and 
spore  thus  give  the  characteristic  drumstick  appearance. 
It  is  easily  stained  with  the  usual  dyes,  and  is  positive  to 
Gram's  method.  Detached  flagella  often  become  massed 
together  in  the  form  of  spirals,  not  unlike  spirochaetes.  In 
specimens  stained  with  watery  solutions  of  gentian-violet 
or  methylene  -  blue,  the  bacillary  protoplasm  stains 
uniformly,  but  any  spores  are  unstained  except  at  the 
periphery,  and  so  look  like  rings.  With  carbol-fuchsin  and 
time,  the  spores  become  uniformly  coloured.  Spores 
may  be  found  free  from  the  bacilli  in  which  they  were 
formed.  The  bacillus  liquefies  gelatin  slowly,  also  blood 
serum,  but  does  not  coagulate  milk.  It  is  a  strict  anaerobe 
(obligatory),  but  can  be  habituated  to  aerobic  life,  though 
with  loss  of  pathogenicity  and  toxin-forming  power.  Grows 
moderately  in  aerobic  conditions  where  other  organisms 
which  use  up  the  oxygen  supply  are  present  (symbiosis). 
Its  growth  is  aided  in  all  media  by  slight  alkalinity, 
presence  of  glucose,  maltose,  or  sodium  formate  (1  to  2  per 
cent),  which  act  as  reducing  agents.  With  carbohydrates 
it  produces  acid.  In  gelatin  and  agar,  moderate  amounts 
of  gas  are  produced,  chiefly  CO  2  ;  but  other  substances 
which  are  volatile  and  cause  a  characteristically  unpleasant 
odour  are  formed.  This  is  described  as  a  peculiar  burnt 
odour,  and  as  that  of  putrefying  organic  matter,  and  is 
said  to  be  largely  due  to  H2S  and  CH3.SH  (methyl 
mercaptan). 

Cultures.— In  deep  glucose  gelatin  stab :  growth  begins 


SPORING    BACILLI  313 

one  inch  or  so  below  the  surface,  in  fine  straight  threads, 
radiating  from  the  needle  track.  Slow  liquefaction,  with 
slight  gas  formation,  takes  place. 

In  agar  stab  (glucose  agar) :  the  growth  is  somewhat 
similar: 

In  glucose  broth :  slight  clouding,  with  later  a  thin 
powdery  deposit  on  the  walls  of  the  tube.  Ordinary  broth 
is  preferred  for  toxin  production. 

On  blood  serum :  growth  with  liquefaction  takes  place. 

In  milk :  acid  is  formed  but  no  clot. 

On  potato :  growth  is  delicate. 

On  agar  plates :  colonies  show  a  compact  centre,  with 
loose  feathery  outline  not  unlike  B.  subtilis  or  anthrax. 

Spores— Resist  dry  heat  at  8o°  C.  for  I  hour ;  live  steam 
for  5  minutes  ;  5  per  cent  acid  carbolic  for  12  to  15  hours  ; 
1  per  cent  corrosive  sublimate  for  2  to  3  hours.  Direct 
sunlight  diminishes  their  virulence  and  ultimately  destroys 
them,  otherwise  they  may  remain  virulent  for  years 
(in  one  case,  11  years).  Are  best  formed  at  370  C,  but 
also  form  at  200  C.  in  8  to  10  days. 

Habitat. — Soil,  street  dust,  horse-dung. 

Pathogenesis. — In  man  :  mostly  from  punctured  wounds. 
The  bacillus  remains  at  the  local  site,  but  the  toxins  are 
carried  to  the  nerve  cells  of  the  motor  horns  of  the  spinal 
cord,  and  of  the  motor  ganglia  of  the  brain.  The  manner 
of  transmission  is  believed  to  be  by  absorption  through 
the  end-plates  of  the  motor  nerves  in  the  muscles,  and 
thence  via  the  axis-cylinder  processes  to  the  respective 
nerve  cell.  The  toxins  have  been  shown  to  have  no  effect 
on  the  motor  or  sensory  endings  of  the  nerves,  but  solely 
as  an  exciter  of  the  nerve  cells  concerned  in  reflex  action 
in  the  cord,  pons,  and  medulla.  The  affinity  of  the  toxins 
for  the  nervous  system  varies  in  different  animals ;  in  the 
guinea-pig  it  is  its  chief  affinity,  whereas  in  the  alligator 
it  shows  no  affinity,  and  intermediate  degrees  exist. 
Section  of  a  nerve,  e.g.,  the  sciatic  nerve,  followed  by 
injection  of  the  toxins  into  the  muscles  supplied  by  that 
nerve,  prevents  the  toxins  reaching  the  spinal  cord ;  but 
if  the  nerve  below  the  section  be  cut  out  and  introduced 
into. a  mouse,  the  animal  will  die  of  tetanus.  Similarly, 
infection  of  one  side  of  the  cord  passes,  when  the  dose  is 


314         PUBLIC    HEALTH    BACTERIOLOGY 

large,  to  the  other  side  via  the  commissure,  and  up  the 
cord  to  the  higher  centres.  The  latter  extension  can  be 
prevented  by  section  of  the  cord.  Lately,  in  India,  the 
relation  between  subcutaneous  or  intramuscular  injections 
of  quinine  (given  for  malaria)  and  the  production  of  tetanus 
has  been  worked  out.  Such  quinine  injections  are  apt  to 
have  a  destructive  action  on  the  tissues,  and  the  foci  of 
dead  tissue  produced  serve  as  suitable  anaerobic  media  for 
the  growth  of  tetanus  spores.  The  latter  are  believed  to 
reach  these  foci  by  absorption  from  the  bowel.  This 
explanation  will  also  probably  serve  for  the  Mulkowal 
outbreak  (1902),  when  19  persons  developed  tetanus  (out  of 
107  injected)  after  inoculations  of  Haffkine's  plague 
prophylactic.  In  India,  tetanus  spores  seem  to  be  present 
in  the  bowel  in  a  considerable  proportion  of  the  natives. 

Toxins. — Broth  cultures  grown  anaerobically  are  usually 
highly  toxic  to  animals,  0-000005  c.c.  (0-00V0  o)  or  ^ess  Demg 
fatal  to  a  mouse  of  10  grm.  weight.  Fatal  dose  for  a  man 
is  given  as  0-23  mgr.,  equal  to  0-003  of  a  grain,  or  ^J^. 
The  maximum  yield  is  given  in  10-  to  14-day-old  cultures. 
After  this  it  rapidly  deteriorates.  This  also  happens  after 
separation  from  the  bacilli  by  filtration,  and  in  a  few  days 
it  may  have  only  TiF  of  its  original  power.  Von  Behring, 
who  first  noted  this  change,  attributed  it  to  the  action  of 
light,  temperature,  and  especially  oxygen,  on  the  toxins ; 
and  so  such  filtrates  should  be  kept,  covered  with  a  layer 
of  toluol  and  in  a  dark  cool  place.  Exposure  for  a  few 
minutes  to  650  C.  destroys  it,  as  do  20  min.  at  6o°  C.  and 
1-5  hour  at  550  C.  Drying  has  no  effect  apart  from 
temperature.  It  can  be  precipitated  by  over-saturation 
of  the  solution  with  ammonium  sulphate,  and  thoroughly 
dried  and  stocked  in  vacuum  tubes,  together  with  anhydrous 
phosphoric  acid,  it  may  be  preserved  indefinitely  without 
deterioration.  The  ordinary  effects  of  the  toxin  are 
attributed  to  "  tetanospasmin."  Besides  this,  a  substance 
named  "  tetanolysin  "  which  has  the  power  of  destroying 
the  red  corpuscles  of  various  animals,  was  discovered 
by  Ehrlich.  Tetanus  toxin  can  be  fully  neutralized  by 
mixing  it  with  brain  substance. 

Tetanus  toxin  is  peculiar  in  that,  after  introduction  into 
an    animal's   body,    a   definite   incubation   period   occurs 


SPORING    BACILLI  315 

before  symptoms  arise.  In  the  guinea-pig  this  is  thirteen 
to  eighteen  hours,  and  in  the  horse  five  days.  It  is  shorter 
after  intravenous  injection,  probably  through  getting  more 
quickly  to  the  nerve  centres.  Crocodiles  are  resistant  to 
tetanus  toxin. 

Immunity.  —  Produced  by  injection  of  filtered  toxin 
in  increasing  doses.  At  Elstree,  the  serum-producing 
department  of  the  Lister  Institute,  London,  the  horse  is 
immunized  by  the  injection  of  0-5  c.c.  filtered  toxin  -f  0-5 
c.c.  of  Lugol's  solution  of  iodine  (1-300),  repeated  at 
intervals  of  ten  days,  gradually  increasing  the  dose  until 
10  c.c.  of  unreduced  toxin  are  given.  The  iodine  solution 
neutralizes  the  toxin  to  some  extent.  The  serum  of  such 
an  immunized  animal  is  antitoxic  ;  but  the  effect  of  its 
injection  into  an  infected  animal  is  not  so  good  as  is  the 
case  with  diphtheria  antitoxin,  because  the  tetanus  is 
mainly  bound  to  the  nervous  tissue  and  is  thus  less 
susceptible  to  the  action  of  the  antitoxin.  Von  Behring 
believes  that  there  is  no  hope  of  its  being  useful  after 
symptoms  have  existed  for  30  hours,  but  MacConkey  and 
Green  say  that  if  much  larger  doses  were  used  better 
results  would  be  got  in  the  human  subject,  comparable  with 
those  reported  in  horses.  The  serum  is  standardized  so 
that  1  grm.  will  protect  100,000,000  grm.  weight  of  mouse 
(v.  Behring),  or  1,000,000,000  grm.  weight  (Pasteur 
Institute).  Of  this  100  c.c.  are  advised  to  be  injected 
subcutaneously,  and  in  the  case  of  the  first,  repeated.  The 
argest  dose  that  can  be  comfortably  given  at  one  spot  is 
20  c.c.  Intravenous  injection  is  said  to  give  better  results 
than  subcutaneous  injection.  The  serum  is  warmed  to 
the  body  temperature  and  slowly  introduced  into  an 
arm  vein,  10  to  20  c.c.  every  few  hours.  Intracerebral 
injection  has  also  been  practised,  but  with  no  better 
results.  Prophylactic  doses  (10  c.c.)  are  advised  as  a 
routine  practice  in  ragged,  bruised,  and  punctured  wounds, 
especially  if  soiled  with  material  likely  to  contain  tetanus 
spores.  The  dose  is  given  without  unnecessary  delay. 
In  U.S.A.,  in  1903,  out  of  4449  Fourth  of  July  accidents, 
406  were  followed  by  death  from  tetanus,  while  in  1907, 
only  62  tetanus  deaths  arose  from  4413  accidents,  and 
much  of  the  decrease  is  attributed  to  the  early  use  of  a 


316         PUBLIC    HEALTH    BACTERIOLOGY 

prophylactic    dose    of    antitoxic    serum.     In    veterinary 
practice,  prophylaxis  has  been  used  with  great  success. 
Search  for  B.  Tetani  in  a  Suspicious  Wound. — 

(a)  Microscopic  examination  of  films    for   drumstick 

shapes. 

(b)  Cultivation  in   deep  stabs  in   glucose   media  for 
forty-eight  hours. 

(c)  Inoculation  into  mice  and  guinea-pigs. 

A  loopful  of  the  discharge  into  the  root  of  the  tail  of  a 
mouse  will  soon  give  rise  to  characteristic  symptoms  if 
B.  tetani  be  present.  From  cultures  Kitasato  uses  splinters 
of  wood  dipped  in  same,  then  heated  to  8o°  C.  for  i  hour, 
which  kills  all  non-sporing  organisms  and  destroys  any 
toxin  developed.  A  splinter  is  introduced  subcutaneously, 
and  if  death  results  it  is  from  the  spores  which  it  carries. 

Bacillus  Botulinus.  —  Found  in  "meat-poisoning" 
by  raw  ham  by  van  Ermengem  in  1896.  The  symptoms 
of  the  illness  resembled  those  following  sausage  (botulus) 
poisoning,  frequently  met  with  in  Germany  from  the 
ingestion  of  raw  sausage.  The  symptoms  follow  at  the 
earliest  in  twelve  to  twenty-four  hours  after  the  eating 
of  the  food.  They  are  due  to  the  action  of  a  soluble  toxin 
on  the  medullary  centres,  causing  dysphagia,  salivation, 
dilated  pupils,  and  respiratory  and  cardiac  distress.  Fever 
is  usually  absent  and  consciousness  is  retained. 

B.  botulinus  is  a  large  bacillus  4  to  9  micra  long  x  0-9  to 
1-2  micron  thick,  with  rounded  ends.  It  is  slightly  motile, 
and  has  four  to  eight  peritrichal  flagella.  It  forms  oval 
spores  at  one  end,  rather  thicker  than  the  bacilli,  and  these 
show  slight  resistance  (1  hour  at  8o°  C).  Strict  anaerobe  ; 
liquefies  gelatin  ;   Gram-positive. 

Cultures. — Characteristic  growth  on  glucose-gelatin 
plates  :  round,  yellowish,  transparent  colonies,  composed 
of  coarse  granules  which  (under  a  low  power)  show  a 
streaming  movement,  especially  at  the  periphery.  Forms 
gas  in  glucose,  but  not  in  lactose  nor  sucrose.  Milk  is  not 
coagulated.  All  cultures  have  a  sour  odour.  The  toxin  is 
closely  related  in  its  action  to  the  toxins  of  diphtheria  and 
tetanus.  The  bacilli  do  not  seem  to  multiply  in  the  body, 
but  the  toxin  is  absorbed  from  the  alimentary  canal  and 
produces  the  symptoms.     The  infected  ham  or  sausage 


SPORING    BACILLI  317 

shows  the  bacilli  in  large  numbers  between  the  fibres. 
Thorough  cooking  destroys  the  toxin  (not  so  in  Gaertner 
meat-poisoning).  The  meat  may  be  without  any  signs 
of  ordinary  decomposition.  An  antitoxin  has  been 
produced  by  Kempner,  who  also  cultivated  the  bacillus 
from  the  intestine  of  the  pig.  Botulism  is  a  dangerous 
affection,  ending  fatally  in  25  per  cent  of  those  attacked. 

Bacillus  of  Malignant  CEdema.  —  Discovered  by 
Pasteur  in  1877,  in  guinea-pigs  inoculated  with  putrefying 
animal  tissues.  Gaffky  found  it  in  the  upper  layers  of 
the  soil  of  gardens  and  in  dust.  It  is  widely  distributed  in 
nature,  and  has  been  found  in  the  intestine  of  animals  and 
man.  Its  spores  are  very  resistant,  and  are  placed  in 
the  centre  or  near  it.  They  are  oval-shaped  and  slightly 
bulge  the  bacterial  body.  Spore  formation  occurs  above 
200  C.  and  is  usually  well  seen  in  forty-eight  hours  at  370  C. 
The  guinea-pig,  rabbit,  sheep,  and  goat  are  susceptible  to 
inoculation  ;  the  ox  is  immune  to  experimental  infection, 
but  has  contracted  the  disease  by  natural  channels.  The 
bacillus  is  long  (4  to  9  micra)  and  rather  thinner  than  the 
anthrax  bacillus,  being  0-9  to  1-2  micron  thick.  The  bacilli 
have  somewhat  rounded  ends,  and  at  times  form  threads. 
They  are  motile,  have  numerous  peritrichal  flagella,  and 
are  strict  anaerobes.  They  stain  readily  by  the  usual 
aniline  dyes ;    they  are  Gram-negative. 

Cultures. — They  grow  best  in  the  presence  of  glucose, 
and  produce  a  heavy,  putrid  odour.  They  liquefy  gelatin, 
and  in  deep  stab  show  bubbles  of  gas  around  the  colonies. 
They  grow  rapidly  in  deep  stab  in  glucose  agar,  and  here 
also  gas  forms  and  usually  splits  the  medium.  In  broth, 
there  is  general  clouding  but  no  pellicle ;  a  granular 
sediment  forms.  In  milk,  slow  coagulation  is  produced. 
On  blood  serum,  growth  is  luxuriant.  On  potato,  growth 
readily  occurs. 

Inoculation  of  a  guinea-pig  (subcutaneously)  produces 
death  in  twenty-four  to  forty-eight  hours.  There  is  an 
intense  inflammatory  oedema  around  the  site  of  puncture 
and  injection,  which  gradually  extends  to  the  surrounding 
tissues.  The  skin  and  subcutaneous  tissues  are  infiltrated 
with  a  reddish-brown  fluid,  are  softened,  contain  bubbles 
of  gas,   and  are  in  places  gangrenous.     The  superficial 


318         PUBLIC    HEALTH    BACTERIOLOGY 

muscles  are  also  involved,  and  have  a  putrid  odour.  The 
internal  organs  are  congested  and  show  parenchymatous 
degeneration.  The  spleen  is  soft,  but  not  much  enlarged. 
Immediately  after  death  bacilli  are  not  found  in  the  blood 
or  internal  organs ;  but  thereafter  the  bacilli  rapidly 
spread  into  the  blood  and  organs.  This  account  applies 
to  the  mixed  infections  (garden  soil)  ;  in  pure  infection, 
little  gas  and  odour  are  formed. 

Toxins. — A  small  amount  of  soluble  toxin  is  formed,  and 
filtrates  of  cultures  in  fluid  media  produce  the  same  sym- 
ptoms (if  used  insufficient  quantity)  as  the  bacilli  themselves. 
Chamberland  and  Roux,  in  1887,  produced  immunity  in 
guinea-pigs  by  the  injection  of  the  toxin,  obtained  by 
filtration  or  by  sterilization  of  cultures  by  heat,  or  by  filtra- 
tion from  the  serum  of  animals  dead  of  the  disease. 

Pasteur  called  the  disease  "  septicemic,"  but  it  is  not 
a  true  septicaemia  like  anthrax,  in  which  the  bacilli  invade 
the  blood  and  organs.  It  is  a  rare  disease,  occurring  in 
man  after  traumatism. 

Quarter-Evil.  —  A  disease  of  cattle,  sheep,  and  goats, 
called  by  the  Germans,  "  Rauschbrand,"  and  by  the 
French,  "  charbon  symptomatique."  It  has  never  been 
observed  in  man.  Infection  takes  place  by  some  wound 
of  the  surface,  and  occasions  inflammatory  swelling  with 
bloody  oedema  and  emphysema  of  the  tissues  ;  the  affected 
part  becomes  greatly  swollen,  and  of  a  dark,  almost  black 
colour.  The  bacillus  is  found  in  the  inflamed  tissues  and 
in  small  numbers  in  the  blood  of  internal  organs.  It 
closely  resembles  the  B.  cedematis  maligni,  but  is  somewhat 
thicker  and  does  not  form  such  long  threads  (filaments). 
The  spores  also  are  more  bulging  and  nearer  the  end  of  the 
bacilli.  An  acute  disease  of  sheep  in  Northern  Europe, 
called  "  braxy,"  is  associated  with  the  presence  of  a  very 
similar,  if  not  identical  anaerobe.  Active  and  passive 
immunization  of  sheep  and  goats  and  cattle  are  practised. 

B.  Enteritidis  Sporogenes  was  first  isolated  by  Klein 
in  1895  from  diarrhoeal  stools.  It  was  afterwards  found  in 
infantile  diarrhoea  and  summer  diarrhoea,  and  as  a  constant 
inhabitant  of  sewage.  It  is  slightly  motile,  with  a  tuft 
of  flagella  at  one  pole  (lophotrichal),  i-6  to  4-8  micra  long 
by  o-8  micron  thick ;  easily  stained  ;  Gram-positive  ;  gelatin- 


SPORING     BACILLI  319 

liquefying,  and  produces  acid  and  gas  in  bile-salt  glucose 
media  and  in  peptone  water  +  glucose  or  mannite.  It 
forms  a  spore  nearer  one  end.  Its  growth  in  milk  is  highly 
characteristic,  and  this  medium  is  commonly  used  for  its 
isolation. 

Method. — A  small  quantity  of  the  suspected  material 
is  inoculated  into  sterile  milk  ("  whole  milk  "),  using  at 
least  15  c.c.  of  the  medium.  Heat  for  10  minutes  at  8o°  C. 
to  destroy  all  non-sporing  forms,  cool  the  tube,  and  incubate 
anaerobically  for  twenty-four  to  thirty-six  hours.  If  the 
casein  is  precipitated  and  torn  into  irregular  masses,  with 
a  moderately  clear  whey  and  abundant  gas  formation, 
the  result  is  positive,  but  it  is  desirable  to  verify  by  animal 
inoculation.  (In  the  examination  of  water  and  milk,  the 
result  is  observed  after  two  days'  incubation.)  The 
culture  has  a  smell  of  butyric  acid,  and  numerous  bacilli 
are  found  in  the  whey.  If  1  c.c.  of  the  whey  be  injected 
into  a  guinea-pig,  the  animal  becomes  ill  in  a  few  hours,  and 
dies  in  twenty-four  hours.  At  the  point  of  inoculation  the 
skin,  subcutaneous  tissues,  and  sometimes  the  adjacent 
muscles,  are  green,  gangrenous,  ill-smelling,  and  cedema- 
tous  ;  there  may  be  gas  formation.  This  pathogenic  test 
serves  to  distinguish  the  B.  enteritidis  sporogenes  from 
the  B.  butyricus  of  Botkin,  which  otherwise  closely 
resembles  it. 

B.  Aerogenes  Capsulatus. — First  observed  by  Welch 
in  1891,  and  obtained  from  the  intravascular  blood  in  a 
case  of  ruptured  aortic  aneurysm.  The  post-mortem  took 
place  six  hours  after  death,  and  attention  was  called  to 
the  blood  by  the  presence  of  gas-bubbles  throughout  the 
vessels.  It  was  fully  described  by  Welch  and  Nuttall  in 
1892,  and  in  1893  Fraenkel  independently  described  (under 
the  name  of  B.  phlegmonis  emphysematosa?)  a  bacillus, 
now  considered  to  be  identical  with  the  B.  aerogenes 
capsulatus.  Klein's  B.  enteritidis  sporogenes  is  believed 
by  some  to  be  the  same  organism,  or  a  closely  related  one. 
B.  aerogenes  capsulatus  is  widely  distributed  in  nature, 
being  found  in  soil,  dust,  brackish  water,  and  in  the 
normal  intestinal  tract  of  man  and  animals.  In  size 
it  is  not  unlike  anthrax  bacillus,  but  is  more  variable  in 
length  and  somewhat  thicker.     The  bacilli  are  generally 


320 


PUBLIC    HEALTH    BACTERIOLOGY 


single  or  in  short  chains,  and  are  shorter  and  thicker  in 
cultures.  Chain  formation  seems  to  occur  in  the  blood 
chiefly,  and  never  in  artificial  culture.  Welch  regards  this 
as  an  important  distinction  from  anthrax  bacilli.  When 
recovered  from  the  body  fluids  it  possesses  a  capsule.  Each 
bacillus  forms  one  spore,  which  may  be  central  or  excentric. 
Anaerobic  ;  non-motile  ;  non-flagellar  ;  Gram-positive  ; 
gelatin-liquefying  (in  most),  and  forming  acid  and  gas  in 
glucose,  lactose,  and  saccharose,  but  not  in  mannite.  In 
milk  the  reaction  is  similar  to  that  described  under  B. 
enteritidis  sporogenes.  It  is  highly  pathogenic  to  guinea- 
pigs  but  not  to  rabbits. 

Isolation. — Make  a  suspension  of  the  suspected  material 
(faeces,  etc.)  in  sterile  salt  solution  (i  c.c.  in  5  c.c). 
Thoroughly  emulsify  and  filter  through  a  sterile  paper, 
and  inject  1  to  2  c.c.  of  the  filtered  suspension  into  the  ear 
vein  of  a  rabbit.  After  5  minutes,  kill  the  rabbit  and 
place  its  dead  body  in  the  incubator  (370  C.)  for  5  or  6  hours. 
At  the  end  of  this  time  the  animal  is  usually  found  tensely 
distended  with  gas,  and  post  mortem  gas  bubbles  will  be 
found  throughout  the  body,  most  characteristically  in  the 
liver,  where  isolated  bubbles  are  found  on  the  surface. 
From  the  bubbles,  smears  and  cultures  may  be  taken. 
Identification  is  made  from  its  morphology,  capsule,  non- 
motility, and  gas  formation.  In  man,  infection  usually 
follows  traumatism.  Distinction  from  B.  enteritidis 
sporogenes :  non-motility,  non-flagellar,  not  fermenting 
mannite.  Muir  and  Ritchie  state  that  it  is  non-gelatin- 
liquefying  and  non-pathogenic  to  guinea-pigs,  but  American 
authors .  describe  it  as  above. 

Summary. 


Bacillus 

Motility 

Flagella 

Gram. 

Gelatin- 
liquefying 

Spore 

Tetani 

+ 

peri- 
trichal 

+ 

+ 

end  (drumstick) 

Botulinus 

+ 

+ 

+ 

near  end 

Malignant  oedema 

+ 

., 

— 

+ 

central 

Quarter-evil 

+ 

" 

— 

+ 

near  end — - 
racket  shape 

Enteritidis     sporo- 

+ 

lopho- 

+ 

+ 

central  or  near 

genes 

trichal 

end 

Aerogenes     capsu- 

— 

— 

+ 

+ 

,,         ,, 

lars       . .      \>   . . 

1 

CHAPTER  XV. 
SPIRILLA. 

SPIRILLUM     CHOLERA     ASIATICS. 

The  cholera  spirillum  was  discovered  by  Koch  in  1883  in 
the  defalcations  of  sufferers  from  cholera.  It  is  also  called 
the  "  comma  bacillus  "  and  the  "  Vibrio  cholerae." 

Description. — Short,  slightly  curved  rods,  1-5  to  2  micra 
long  by  0-5  micron  thick.  Ex.  C  In  pairs,  may  form  an 
S-shape,  thus  *y  Actively  motile,  and  swim  like  fish  in 
lines,  thus  5  5  =  5 .  Possess  one  flagellum,  situated  at  one 
end  (monotrichal) .  Non-sporing  ;  not  phosphorescent  ; 
Gram-negative.  Markedly  aerobic,  but  can  grow  anaero- 
bically.  Optimum  temperature  370  C.  ;  growth  usually 
ceases  at  160  C.  Gelatin-liquefying.  Give  nitroso-indol 
reaction  with  sulphuric  acid  within  twenty-four  hours. 

Culture. — Grows  readily  on  all  usual  media,  but  better  if 
alkaline,  and  except  on  potato  even  at  room  temperature  ; 
characteristic  on  gelatin  plates  and  in  broth.  On  gelatin 
plate :  minute  whitish  points  in  twenty-four  to  forty-eight 
hours ;  surface  when  magnified  is  coarsely  granular  and 
furrowed.  Liquefaction  follows  and  the  colony  sinks, 
showing  a  ring  around.  In  gelatin  stab :  liquefaction 
begins  at  the  surface,  with  gradual  formation  of  a  funnel 
of  liquefaction.  In  broth :  rapid  clouding  occurs  with 
wrinkled  pellicle  on  top,  composed  of  spirilla  in  a  very 
actively  motile  condition.  In  milk :  growth  but  no  visible 
change.  In  peptone  water  :  rapid  growth  with  production 
of  indol,  and  reduction  of  nitrate  to  nitrite,  hence  a  few 
drops  of  pure  sulphuric  acid  will  give  a  red  colour — the 
so-called  cholera-red  reaction.  Also  given  in  broth  culture, 
and  in  both  in  twenty-four  hours,  owing  to  rapid  growth. 
Not  absolutely  specific,  as  it  is  given  also  by  Sp.  Metch- 
nikovi.  Blood  serum  is  rapidly  liquefied.  In  sugar  media  : 
no  gas  is  formed,  but  acid  with  glucose.  Does  not  pro- 
duce haemolysis,  though  very  similar  species  do.  Does  not 
multiply  in  water. 

21 


322  PUBLIC    HEALTH    BACTERIOLOGY 

Staining. — Readily  with  usual  stains,  best  with  Loeffler's 
methylene-blue  and  weak  carbol-fuchsin.  Loses  stain  by 
Gram's  method. 

Resistance. — Not  great.  Killed  in  ten  minutes  at  6o°  C.  ; 
on  drying,  in  two  hours.  Mineral  acids,  I  in  5000  to  1  in 
10,000,  destroy  it  in  a  few  minutes.  The  gastric  juice  con- 
tains 2  parts  of  HC1  per  1000 ;  hence  it  is  killed  by  gastric 
juice,  but  can  flourish  in  intestine.  Freezing  kills  it  in 
three  to  four  days. 

Agglutination — Is  shown  by  serum  of  cholera  con- 
valescents in  dilutions  of  1-15  to  1-120.  Present  eight  to 
ten  days  after  attack,  most  marked  twenty-eight  days 
after,  and  gradually  diminishes.  Has  been  noted  as  early 
as  first  day  of  disease. 

Pathogenesis. — For  man :  has  been  established  by 
laboratory  accidents.  Does  not  invade  blood  ;  immense 
numbers  in  stools  ;  in  rice-water  stools,  loosened  epithelial 
cells  loaded  with  vibrios.  Not  in  urine.  Infection  by 
mouth.  Disappears  from  stools  in  two  to  three  days. 
Cholera  carriers :  healthy  persons  whose  faeces  contain 
virulent  cholera  spirilla.  Mostly  spread  by  water,  fomites, 
ringers,  flies.  For  animals  :  not  established,  though  vibrios 
on  teats  of  suckling  mother  have  infected  young,  with 
choleraic  symptoms.  But  as  this  result  or  similar  ones  have 
been  given  by  other  vibrios,  specificity  cannot  be  founded 
on  this  test.  Intraperitoneal  injection  in  guinea-pigs  is 
followed  by  general  symptoms,  with  abdominal  distension, 
subnormal  temperature,  and  profound  collapse.  There  is 
peritoneal  effusion  which  may  be  almost  clear,  or  with 
flakes  of  lymph.  There  is  little  tendency  to  invade  the 
blood-stream,  and  the  symptoms  are  mainly  due  to  an 
intoxication.     It  is  not  pathogenic  to  pigeons. 

Toxins. — Filtered  cholera  cultures  have  as  a  rule  little 
toxic  action  ;  hence  it  is  inferred  that  little  soluble  toxin 
is  formed,  but  mostly  endotoxin.  Results  at  present  are 
conflicting. 

Pfeiffer's  Reaction. — If  cholera  spirilla  are  injected  into 
the  peritoneum  of  an  immunized  guinea-pig,  they  first 
lose  their  motility,  then  swell  up  and  crumble  into  frag- 
ments, which  finally  melt  away  and  disappear.  This 
lysis  is  also  manifested  in  a  test  tube  in  a  mixture  of  serum 


SPIRILLA  323 

plus  vibrios.  Or,  inject  a  mixture  of  one  loopful  (2  mgr. 
of  recent  agar  culture  and  1  c.c.  of  broth  containing  o-ooi 
c.c.  of  anti-cholera  serum  into  the  peritoneum  of  a  guinea- 
pig.  Remove  some  fluid  at  regular  intervals  and  examine. 
If  above  reaction  is  got,  then  said  to  be  positive,  and  in  case 
described,  organism  is  proved  to  be  true  cholera  spirillum. 
If  the  latter  is  used  against  an  unknown  serum,  then  the 
anti-power  of  the  serum  can  be  determined  and,  if  positive, 
by  using  various  dilutions,  the  bacteriolytic  power. 

Immunization. — A  guinea-pig  is  easily  immunized  by 
repeated  injections  of  non-fatal  doses  of  dead  spirilla  ; 
later  small  doses  of  living  organisms  may  be  used.  A  high 
degree  of  immunity  is  thus  developed,  and  the  blood 
serum  of  such  an  animal  (anti-cholera  serum)  when 
injected  into  another  guinea-pig  has  marked  protective 
power.  This  is  not  due  to  any  antitoxic  substances,  but 
to  antibacterial  power.  Cholera-immune  serum  is  thus 
bacteriolytic,  not  antitoxic.  This  power  is  specific,  and 
does  not  apply  to  other  closely  related  organisms. 

Hafjkine's  Vaccine. — First  vaccine  :  Attenuated  virus 
by  long  cultivation  at  390  C.  or  by  other  methods. 
Second  vaccine  :  Given  five  days  later,  of  virulent  virus 
(by  passage  through  guinea-pigs).  Both  given  sub- 
cutaneously.  Lately  has  given  only  one,  and  that  the 
"  virus  exalte."  General  conclusions  as  to  efficacy : 
(1)  Protective  effect  of  anti-cholera  vaccine  commences 
soon  after  operation  and  increases  rapidly  for  first  four 
days,  and  lasts  fourteen  months,  after  which  it  diminishes 
and  completely  disappears.  Larger  doses  cause  longer 
effects.  (2)  During  period  of  its  activity,  the  number  of 
cases  among  vaccinated  is  one-tenth  of  number  among 
others.  (3)  Mortality  among  those  attacked  differs  but 
little,  and  the  course  of  the  disease  is  not  affected  by 
the  previous  inoculation. 

Isolation.— 

1.  From  Faces. — Make  pre-culture  in  peptone  water. 
(a)  Inoculate  peptone  water  in  Erlenmeyer  flasks  (1  loopful 
in  each),  (b)  In  eight  hours,  if  a  film  appears  or  not, 
make  hanging  drop  from  surface,  and  if  you  get  fish  trains, 
dry  and  stain  to  see  if  form  is  typical,  (c)  Subculture 
from  film  on  gelatin  plates,  and  smear  over  agar  plates. 


324         PUBLIC    HEALTH    BACTERIOLOGY 

(d)  After  eight  to  ten  hours  examine  any  colonies,  and  if 
pure  cultures,  plant  out  as  follows  :  (i)  Peptone  and  salt 
solution :  in  twenty-four  hours  at  370  C,  turbid,  and  gives 
cholera-red ;  (ii)  Gelatin  plates  :  characteristic  colonies 
with  irregular  margins  ;  (hi)  Gelatin  stab  :  typical  funnel- 
shaped  liquefaction ;  (iv)  Agar  slope  :  growth  in  twenty- 
four  hours  at  370  C.  must  give  with  anti-cholera  serum, 
agglutination  and  Pfeiffer's  test  ;  (v)  A  portion  of  colony 
should  be  examined  for  typical  microscopical  appearances. 

Make  films  and  hanging  drops  direct  from  stools. 
Dunbar  diagnoses  from  two  hanging  drops,  one  having 
added  to  it  an  equal  quantity  of  1-50  normal  serum,  the 
other  an  equal  quantity  of  1-500  anti-cholera  serum. 
Cholera  organisms  retain  their  motility  in  the  first  instance, 
but  lose  it  and  agglutinate  in  the  second.  The  hanging 
drop  is  mounted  from  peptone  water  in  which  a  piece  of 
mucus  has  been  broken  up. 

2.  From  Water. — Keep  10  per  cent  peptone  water 
sterilized.  Take  900  c.c.  of  suspected  water,  and  add 
100  c.c.  of  strong  peptone  water.  Divide  into  ten  flasks, 
each  containing  100  c.c.  Incubate  at  370  C.  In  eight  to 
twelve  hours  make  a  film  and  hanging-drop  preparation 
from  the  surface  of  each  flask.  From  those  flasks  showing 
most  similar  forms,  make  subcultures,  proceeding  as 
above.  If  a  spirillum  conforms  to  all  the  above  tests,  it  is 
probably  the  true  cholera  vibrio,  but  it  must  be  remem- 
bered that  a  certain  number  of  spirilla,  although  agglutina- 
ting to  some  extent  with  cholera  serum,  are  sharply 
differentiated  by  being  multiciliated  and  hemolytic. 


SPIRILLA    OTHER    THAN     THE 

CHOLERA    SPIRILLUM. 

(Often  present  in  water  but  not  necessarily  pathogenic). 

Sp.  Metchnikovi — Is  found  in  a  disease  resembling  fowl 
cholera  in  the  faeces  and  blood.  It  is  practically  identical 
with  the  Sp.  cholerae,  having  a  single  polar  flagellum. 
Culturally,  it  fluidifies  gelatin  twice  as  rapidly,  and  grows 
slightly  more  luxuriantly.  It  is  sharply  distinguished, 
however,   by  being  very  pathogenic  to  pigeons   (doves), 


SPIRILLA  325 

whereas  Sp.  cholerae  is  scarcely  so.  It  is  negative  to 
Pfeiffer's  test,  but  gives  the  cholera-red  reaction. 

Sp.  Massaua  (Massowah) — Was  isolated  in  a  small 
epidemic  of  cholera  and  accepted  as  the  true  spirillum, 
but  further  study  showed  that  it  was  negative  to  Pfeiffer, 
was  very  pathogenic  to  pigeons,  and  possessed  four  flagella. 

Sp.  of  Finkler  and  Prior— Was  isolated  first  from 
faeces  of  a  case  of  cholera  nostras,  and  has  since  been  found 
in  water.  Morphologically,  it  is  like  the  Sp.  cholerae, 
though  thicker  in  the  centre  and  more  pointed  at  the  ends. 
It  does  not  give  the  cholera-red  reaction,  and  liquefies 
gelatin  very  rapidly,  showing  no  bubble-like  appearance. 
Grows  well  on  potato,  and  is  negative  to  Pfeiffer. 

Sp.  Aquatilis  of  Gunther — Was  found  in  Spree  water, 
and  closely  resembles  Sp.  cholerae,  but  young  colonies 
have  a  smooth  rim,  and  it  does  not  give  cholera-red 
reaction,  and  is  negative  to  Pfeiffer.  Does  not  grow  on 
potato. 

Sp.  Danubicus. — Cultivated  from  canal  water.  Does 
not  give  Pfeiffer,  and  colonies  are  different ;  otherwise  it 
closely  resembles  the  cholera  spirillum. 

Sp.  Deneke — Also  called  Sp.  tyrogenum,  was  isolated 
from  butter  and  old  cheese.  It  closely  resembles  the  Sp. 
cholerae,  but  is  thinner  and  smaller ;  growth  in  gelatin 
similar  but  more  rapid,  and  does  not  give  the  cholera-red 
reaction.  It  is  very  feebly  pathogenic,  and  is  usually 
regarded  as  a  harmless  saprophyte. 

Sp.  Phosphorescens — Gives  luminous  cultures. 


CHAPTER     XVI. 

SPIROCHETES. 

The  diseases  produced  by  spirochetes  are  now  referred 
to  as  spirilloses  or  spirochetoses,  and  fall  into  (following 
Muir  and  Ritchie)  two  main  groups  : — (i)  The  human 
spirillar  fevers,  and  the  corresponding  affections  of  various 
animals  ;  (2)  Syphilis  and  yaws,  and  the  ulcerative  and 
gangrenous  conditions  apparently  caused  by  spirochetes 
(e.g.,  Vincent's  angina).  In  the  first  group,  blood  infec- 
tion is  the  rule,  and  the  organisms  are,  in  most  cases  if 
not  in  all,  transmitted  by  blood-sucking  ecto-parasites. 
In  the  second  group,  the  organisms  are  primarily  tissue 
parasites,  and  later  show  blood  infection,  and  are  mainly 
spread  by  direct  contact.  As  regards  general  morphology, 
staining  reactions,  and  conditions  of  growth  and  culture, 
the  various  spirochetes  present  common  characters,  and 
their  classification  with  bacteria  or  protozoa  is  still  a 
matter  of  doubt. 

Spirillum  Obermeieri  or  Sp.  of  relapsing  fever  (Ober- 
meyer,  1873) — Is  now  usually  regarded  as  a  spirochete,  and 
known  as  the  Spirochaeta  recurrentis.  It  is  found  in  the 
blood  of  patients  suffering  from  "  relapsing  fever  "  from 
shortly  before  the  onset  of  the  pyrexia  until  shortly  before 
the  crisis,  and  similarly  in  the  relapses.  The  relationship 
of  the  organism  to  the  disease  has  been  proved  by  the 
injection  of  spirochetes  into  the  blood-stream  causing 
the  typical  attack,  both  in  the  human  subject  and  in 
monkeys  ;  also  in  white  mice  and  rats,  but  in  these  and 
in  monkeys,  relapse  is  rare.  Sp.  Obermeieri  is  a  delicate 
spiral  thread,  7  to  9  micra  long  by  about  1  micron  thick, 
but  the  size  varies  from  one-half  to  nine  times  the  diameter 
of  a  red  blood  corpuscle  (7  micra).  The  windings  like- 
wise vary  from  4  to  10  or  more.  It  stains  with  watery 
basic  aniline  dyes,  somewhat  faintly,  but  best  with  the 
Romanowsky  stains.     It  shows  a  homogeneous  cell  body 


SPIROCHETES  327 

or  a  few  granules,  but  no  division  into  segments.      It  is 
Gram-negative.     Flagella  have  been  noted. 

Spirochaeta  Vincenti— Is  a  delicate  spiral-shaped 
organism,  said  to  be  often  found  in  the  mouth  as  a  simple 
saprophyte.  It  was  described  by  Vincent  in  1896,  in  an 
inflammatory  lesion  of  the  pharynx,  since  spoken  of  as 
Vincent's  angina,  in  association  with  a  fusiform  bacillus 
of  large  size  (3  to  10  micra  by  0-5  to  o-8  micron).  The 
curvature  of  the  spirochetes  is  irregular  and  the  number 
of  curves  variable.  The  relationship  of  these  organisms  to 
the  disease  is  still  obscure. 


THE     MICRO-ORGANISM     OF     SYPHILIS. 

This  is  variously  called  the  Spirochaeta  pallida  of 
Schaudinn  and  Hoffmann,  the  Treponema  pallidum,  and 
the  Microspironema  pallidum.  It  was  discovered  by  the 
two  observers  named  in  1905,  in  the  primary  sore  and 
in  the  adjacent  lymphatic  glands.  It  has  since  been 
demonstrated  in  numerous  lesions  and  in  the  blood,  and 
in  congenital  syphilis.  It  has  been  found  in  large 
numbers  and  in  pure  culture  in  the  lungs,  liver,  spleen, 
pancreas,  and  kidneys  ;  and  in  a  few  cases  in  the  heart 
muscle.  It  has  also  been  found  in  the  roseolar  spots  in 
the  disease,  and  in  blister  fluid  in  an  infected  person. 
This  shows  how  the  disease  can  be  spread  (as  at  times 
it  has   been)  by  vaccine  fluid. 

In  the  bubo,  other  spirochaetes  are  usually  found  in 
association  with  the  Spirochaeta  pallida.  These  are 
much  thicker,  less  undulated,  more  retractile,  and  more 
deeply  staining.  They  are  spoken  of  as  Spirochaeta 
refringens.  These  are  said  to  have  an  undulating  membrane 
(like  the  trypanosomes)  but  no  flagella.  The  term 
treponema  is  now  reserved  for  a  genus  with  no  undulating 
membrane,  and  flagella  of  some  sort  at  their  extremities. 
To  this  group  the  organism  of  syphilis  belongs,  and  is 
hence  now  referred  to  more  strictly  as  the  Treponema 
pallidum. 

The  Treponema  pallidum  is  an  extremely  delicate 
spiral  organism,  very  slender,  about  6  to  14  micra  long  by 
less  than  0-5  micron  thick,  showing  about  a  dozen  very 


328         PUBLIC    HEALTH    BACTERIOLOGY 

regular  spiral  turns  close  together,  the  whole  resembling 
a  fine  corkscrew.  It  has  a  flagellum  at  each  extremity, 
but  no  undulating  membrane.  It  multiplies  by  longi- 
tudinal division,  the  initial  stage  being  shown  by  the 
splitting  of  the  flagellum  at  one  end.  It  can  be  demon- 
strated in  the  living  state  in  a  hanging  drop,  or  a  ringed-in 
cover-slip,  cutting  down  the  light  to  a  minimum,  or  better, 
by  using  dark-field  illumination.  In  smears,  it  can  be 
stained  by  Giemsa's  method,  of  which  there  are  several 
modifications.  A  more  rapid  and  simple  method  is  by 
using  India  ink.  A  loopful  of  secretion  from  a  chancre  is 
mixed  with  a  loopful  of  ink  (Gunther  &  Wagner's  liquid 
pearl  ink),  and  the  mixture  made  into  a  smear  as  for  blood. 
Dry  in  the  air,  and  examine  with  an  oil-immersion  lens. 
The  treponemata  appear  as  white  spirals  on  a  dark  back- 
ground. In  tissues,  ordinary  methods  do  not  stain  the 
organisms.  Levaditi's  method  is  commonly  used,  and 
consists  in  fixing  in  formalin  for  24  hours,  washing  out  the 
formalin  with  water,  the  traces  of  which,  which  might  con- 
tain formalin,  being  removed  with  alcohol ;  soak  in  silver 
nitrate  solution  for  from  three  to  five  days,  wash  and  soak 
in  pyrogallol-formalin  solution  ;  wash,  dehydrate,  imbed  in 
paraffin,  and  section.  A  shorter  method  has  been  devised. 
The  evidence  of  its  pathogenicity  is  derived  from  its 
constant  presence  in  the  lesions  of  acquired  and  congenital 
syphilis,  and  in  that  it  has  been  communicated  to  monkeys, 
producing  typical  syphilitic  course  and  lesions,  from  which 
the  treponema  was  recovered  in  70  per  cent  of  the  cases 
examined.  It  has  not  yet  been  successfully  cultivated  in 
a  pure  state,  or  in  that  case  complement  fixation  by  a 
pure  culture  might  be  an  additional  proof.  It  is  not 
regularly  recovered  from  tertiary  lesions,  which  is  not 
surprising.  This  is  analogous  to  tuberculosis,  in  which 
the  tubercle  bacillus  is  often  not  demonstrable  by  ordinary 
methods  in  the  chronic  lesions.  Treponema  pallidum 
does  not  pass  through  a  filter. 

Wassermann  Reaction — Is  now  regularly  used  in  clinical 
diagnosis.  It  is  described  on  page  205  ;  its  value  is 
discussed  on  page  209.  In  a  recent  research  by  Calmette, 
Breton,  and  Couvrer,  it  has  been  practically  applied 
to   the  diagnosis    of  syphilis    in   the    newly  born   child, 


SPIROCHETES  329 

with  a  view  to  securing  early  treatment  in  those  cases 
which  would  not  be  diagnosed  on  ordinary  clinical 
grounds.  The  mode  of  procedure  was  to  examine  the 
placental  blood  taken  at  the  time  of  delivery,  before 
ligature  of  the  cord.  Out  of  103  such  samples,  in  16  a 
positive  result  was  obtained.  Out  of  the  16,  no  evidence 
was  got  of  syphilis  by  clinical  examination  or  history  in 
the  parents  or  child  in  8  cases.  Of  the  remaining  8,  there 
was  definite  evidence  in  the  parents  or  child  in  5  cases  ; 
and  in  one  other  the  mother  had  previously  borne  an 
anencephalic  monster.  In  every  case  in  which  the  child 
showed  signs  of  syphilis  at  birth,  the  mother's  blood  gave 
a  positive  reaction. 

Immunization. — Though  one  attack  of  syphilis  usually 
protects  against  another  infection  beginning  with  a  chancre, 
no  success  has  yet  been  obtained  in  attempts  to  produce 
either  active  or  passive  immunization. 

Yaws. — A  disease  resembling  syphilis,  and  at  times 
regarded  as  identical  with  it,  which  prevails  endemically  in 
the  West  Indies,  Brazil,  Fiji,  Ceylon,  the  East  Indies,  and 
different  parts  of  Africa.  It  is  called  variously  yaws,  fram- 
bcesia,  bubas,  koko,  paranghi,  and  pian.  It  is  highly  con- 
tagious, but  is  not  hereditary  or  congenital.  It  is  now 
believed  to  be  due  to  Treponema  pertenue,  first  described  by 
Castellani  in  1905  as  Spirochaeta  pertenuis,  which  resembles 
the  Treponema  pallidum  closely. 


CHAPTER    XVII. 

YEASTS     AND     MOULDS. 

Yeasts  and  moulds  are  grouped  in  the  class  of  fungi  to 
which  the  bacteria  also  belong.  They  are  distinguished 
from  the  latter  (and  from  one  another)  by  their  mode  of 
reproduction.  From  the  bacteria  they  also  differ  in  being 
much  larger,  as  a  rule.  Their  biological  requirements 
are  also,  generally,  much  less  exacting. 

Between  these  groups  and  the  average  bacterium,  the 
space  is  bridged  by  some  forms  called  the  higher  bacteria, 
which  resemble  the  moulds  in  showing  branching.  Such 
are  actinomyces  (which  has  been  considered  immediately 
after  B.  tuberculosis),  the  streptothricae,  etc.  These  are 
often  grouped  as  trichomycetes,  which  is  regarded  as  a 
subdivision  of  the  true  moulds.  The  whole  subject  is  at 
present  uncertain  and  confused.  Foulerton,  in  his  Milroy 
Lectures  (Lancet,  1910,  Vol.  i.,  p.  551  on)  urges  the  view 
that  the  micro-organisms  variously  called  tubercle  bacilli, 
actinomyces,  cladothrix  nocardia,  oospora,  and  strepto- 
thrix,  belong  to  one  family  of  moulds  or  hyphomycetes. 

In  clinical  medicine  and  pathology  the  term  "  mycoses  " 
is  used  (following  Virchow)  to  denote  all  the  affections 
produced  by  filamentous  and  budding  fungi,  and  this  term 
associated  with  the  seat  of  the  lesion  has  given  rise  to  such 
terms  as  dermatomycosis,  otomycosis,  etc.  On  the  other 
hand,  such  terms  as  actinomycosis,  saccharomycosis, 
blastomycosis,  aspergillosis,  sporotrichosis,  etc.,  are  used. 

All  the  members  of  these  groups  may  be  considered  as 
facultative  parasites,  parasitic  life  being  unnecessary  to 
their  cycle  of  evolution,  as  their  proper  existence  is  a 
saprophytic  one.  The  parasitism  is,  in  their  case,  simply 
a  phenomenon  of  adaptation. 

Being  destitute  of  chlorophyll,  they  do  not  need  light, 
and  grow  luxuriantly  in  the  dark.  They  vary  much  in 
their  temperature  requirements,   a  few  growing  well  at 


YEASTS    AND    MOULDS  331 

human  body-heat,  some  at  300  to  330  C,  and  most  at  air 
temperatures.  Growth  at  temperatures  higher  than  the 
optimum,  in  certain  media  and  anaerobically,  results  in  the 
production  of  pleomorphic  forms.  Most  of  them  die  when 
deprived  of  air  or  oxygen  ;  a  few  are  anaerobic.  Moisture 
is  absolutely  necessary.  They  grow  readily  on  organic 
matter  of  all  kinds.  The  natural  media  commonly  used 
in  their  study  are  bread,  sterilized  milk,  beer  wort,  potato, 
carrot,  decoctions  of  fruits,  etc.  As  these  media  are  vari- 
able in  their  composition  from  time  to  time,  and  the 
problem  of  pleomorphism  has  to  be  faced,  artificial  media 
of  definite  composition  and  reaction  are  preferred  in 
scientific  work  for  giving  comparable  results.  Growth  is 
best  on  solid  media,  standardized  to  an  acid  reaction  of 
-f  2  per  cent  (+20  per  litre). 


YEASTS. 

The  yeasts  are  fungi  characterized  by  the  mode  of 
multiplication  known  as  "  budding  "  or  "  gemmation' '  or 
asymmetrical  fission,  and  are  hence  called  blastomycetes. 
From  their  action  in  fermenting  sugars  they  have  also  been 
called  saccharomycetes.  Their  botanical  position  as  a 
separate  group  is  not  well  established,  as  a  large  number  of 
intermediate  forms  relate  them  closely  to  the  moulds. 
The  usual  yeast  cell  is  round  or  oval  in  shape,  10  to  20  micra 
long  by  5  to  15  micra  across,  and  occurs  singly  or  in  short 
chains.  Each  cell  is  bounded  by  a  cell-membrane  composed 
of  cellulose,  and  of  such  a  thickness  (0-5  micron)  that  it 
shows  a  double  contour.  Within  the  membrane  is  con- 
tained the  protoplasm,  in  which  is  a  large  number  of 
granules,  globules,  and  vacuoles,  and  in  old  cultures  a 
nucleus  is  sometimes  seen.  When  budding,  the  mother  cell 
throws  out  a  small  globular  process,  which  gradually 
enlarges  until  it  attains  nearly  the  same  size  as  the  parent 
cell.  By  a  gradual  narrowing  of  the  isthmus  between  the 
mother  and  daughter  cells,  the  daughter  cell  finally  becomes 
free.  In  addition  to  this  mode  of  reproduction,  most  yeasts 
can  form  spores  called  "  ascospores."  This  takes  place 
when  there  is  a  lack  of  nourishment  or  where  the  conditions 
of    life    are    otherwise    unfavourable.     These    spores    are 


332         PUBLIC     HEALTH    BACTERIOLOGY 

formed  by  endogenous  cell-division,  and  the  usual  rule 
is  for  the  protoplasm  of  one  cell  to  divide  into  four  spores, 
each  with  its  own  cell-membrane,  the  original  cell- 
membrane  persisting  and  serving  as  an  envelope  enclosing 
the  spores. 

Yeasts  grow  more  slowly  than  bacteria,  and  are  hence 
more  difficult  to  isolate  from  mixed  cultures  on  the 
ordinary  media.  Once  isolated  they  are  kept  alive  by 
subculture  every  2  to  3  months.  On  glucose  agar  or  plain 
agar,  colonies  appear  in  3  to  4  days  as  minute  glistening 
white  spots.  In  stab,  the  growth  is  all  at  the  top,  forming 
a  heaped-up  creamy  layer  on  the  surface  of  the  medium. 
In  broth,  a  stringy  gelatinous  growth  is  formed.  Growth 
takes  place  on  gelatin,  which  is  not  liquefied.  On  potato, 
growth  is  more  rapid.  The  cultivated  yeasts  used  in 
brewing  and  baking  processes  are  capable  of  fermenting 
various  sugars.  This  action  is  due  to  certain  ferments  or 
enzymes  elaborated  by  the  yeasts.  Two  of  the  enzymes 
are  diffused  into  the  medium  in  which  growth  is  taking 
place  ;  one  remains  closely  bound  to  the  yeast  cell,  and 
was  only  isolated  by  Buchner  by  rupturing  the  living 
yeast  cells  under  great  pressure,  filtering,  and  centrifugal- 
izing  the  filtrate.  This  filtrate  was  found  to  have  the 
power  of  fermenting  glucose  and  laevulose  into  alcohol 
and  carbonic  acid  gas.  This  endo-enzyme  is  called 
"  zymase." 

C6H1206  =  2-C2H,-OH  +>C02 

The  other  two  soluble  enzymes  are  "  invertase "  and 
"  maltase."  The  former  inverts  cane  sugar  or  saccharose 
into  invert  sugar  (glucose  -f-  laevulose),  and  so  renders  it 
susceptible  to  the  action  of  the  "  zymase."  "  Maltase  " 
acts  on  malt  sugar,  changing  it  into  glucose,  which  is  then 
acted  on  by  the  "  zymase." 

C,,H22Ou  +  H20  =  C6H1206  +  C6HH06 
Saccharose.  Glucose  -f-  laevulose. 

ClaHMOn  +  H20  =  C„HlaOa  +  QHl206 
Maltose.  Glucose  -f-  glucose. 

Saccharomvces  cerevisise. — This  is  the  name  applied 
to  the  yeast  in  common  use  by  brewers  and  bakers. 


YEASTS     AND     MOULDS  333 

It  consists  of  round  or  oval  cells  containing  a  clear  fluid 
and  no  granules.  It  is  not  used  older  than  one  week's 
growth,  after  which  time  granules  appear  in  the  cells. 

When  budding  very  rapidly,  delicate  mycelial  threads 
are  formed.  It  forms  ascospores  at  25 °  C.  in  thirty  hours, 
and  at  120  C.  in  ten  days.  In  brewing,  yeast  is  added  to 
the  beer  wort  (cooled  to  160  C),  and  fermentation  takes 
place.  A  brownish-yellow  scum  forms  on  the  surface, 
bubbles  of  gas  (C02)  escape,  forming  a  foam,  and  alcohol 
is  formed  in  the  liquid.  This  is  the  "  high  "  fermentation 
with  "  top  "  yeast,  and  takes  place  in  several  days. 

In  some  breweries  "  low  "  or  "  bottom  "  yeast  is  used, 
and  the  fermentation  is  conducted  at  50  C.  The  yeast  cells 
sink  to  the  bottom  as  they  are  formed,  and  the  whole 
process  takes  a  much  longer  time  (fourteen  days). 

In  baking,  the  yeast  converts  the  starch  into  sugar,  and 
then  the  latter  into  C02  and  alcohol.  The  gas  breaks 
up  the  glutin  into  thin-walled  cells.  The  subsequent 
"  baking"  or  heating  kills  the  ferment,  and  drives  off  the 
CO  2  and  alcohol. 

Wild  yeasts  are  usually  termed  torulcc.  They  have 
oval  or  spherical-shaped  cells,  do  not  produce  ascospores, 
and  have  only  feeble  fermentative  powers.  Some  of  them 
have  been  found  to  produce  a  true  mycelium. 

Torula  rosea  (Saccharomyces  rosaceus)  is  a  pink  torula, 
which,  growing  on  gelatin,  agar,  etc.,  produces  raised 
masses  with  a  polished  pink  surface,  similar  to  a  piece  of 
coral.  Microscopically,  it  shows  rounded  or  slightly  oval 
cells,  5  to  8  micra  in  diameter,  and  containing  a  delicate 
yellow  pigment,  which  in  the  mass  gives  the  pink  shade. 

Torula  niger  (Saccharomyces  niger)  grows  on  gelatin  as  a 
black  heaped-up  mass,  resembling  a  piece  of  black  sealing- 
wax.  On  potato  and  bread  paste  it  forms  a  dull  sooty 
crust,  and  in  milk  a  black  crust.     It  is  met  with  in  the  air. 

Pathogenic  yeasts  have  been  described  in  connection 
with  (1)  Multiple  abscesses  in  bones,  lungs,  spleen,  and 
kidney,  and  ending  fatally  (Busse)  ;  (2)  An  illness  simula- 
ting diphtheria  (Klein  and  Gordon)  ;  (3)  Subcutaneous 
myxomatous  tumours  (Curtis)  ;  (4)  Middle-ear  disease 
(Maggiora  and  Gradenigo)  ;   (5)  A  lupus-like  skin  disease 


834         PUBLIC    HEALTH    BACTERIOLOGY 

(Gilchrist)  ;    (6)    An    intraperitoneal  tumour   (Blanchard, 
Schwartz,   and  Binot),   etc. 


MOULDS. 

The  distinguishing  feature  of  the  moulds  is  their  growth 
in  long  threads  or  filaments,  with  seed-bearing  branches 
called  hyphae.  Each  filament  may  be  a  single,  simple, 
multinuclear  cell,  or  a  greatly  branched  one,  or  may  be 
composed  of  a  row  of  cells  set  end  to  end.  The  interlacing 
mass  of  threads  is  called  the  "  mycelium."  On  this  basis 
moulds  are  divided  into  two  classes  :  (i)  Phycomycetes, 
or  those  in  which  the  mycelial  threads  consist  of  a  single 
cell ;  and  (2)  Mycomycetes,  in  which  the  mycelial  threads 
are  composed  of  numerous  cells.  The  two  groups  also 
differ  in  that  in  the  first,  reproduction  is  sexual  and  asexual ; 
and  in  the  second  is  by  the  asexual  process  only.  All 
moulds  prefer  an  acid  to  an  alkaline  medium,  and  hence 
are  found  attacking  fruit  preserves  and  similar  substances. 
The  spores  of  moulds  are  present  everywhere,  and  in 
the  air  are  in  greater  numbers  than  bacteria. 

The  members  of  this  group  of  fungi  which  we  shall 
consider  are  :  Mucor  mucedo  ;  Aspergillus  ;  Penicillium ; 
Microsporon  furfur ;  M.  minutissimum ;  Sporotrichum 
Beurmanni ;  Oidium  albicans ;  and  the  moulds  of  ring- 
worm. 

Mucor  mucedo  is  the  commonest  mucor  or  "head" 
mould,  and  belongs  to  the  class  of  single-celled  fungi  or 
phycomycetes.  It  is  the  common,  white,  cottony  mould 
which  grows  on  damp  bread,  rotten  fruit,  horse  dung,  etc. 
There  is  a  finely  branched  mycelium  from  which  project 
thicker  unbranched  hyphae.  Near  the  end  of  these  hyphae 
a  septum  forms,  the  terminal  portion  of  the  hypha  swells, 
and  in  it  numerous  oval  spores  develop.  The  globular 
swelling  produced  is  known  as  the  "  sporangium,"  and 
is  enclosed  by  a  capsule.  It  ruptures  when  ripe  by  the 
swelling  of  the  gelatinous  material  in  which  the  spores  are 
imbedded.  The  end  of  the  hypha  projects  into  the  spor- 
angium, and  this  part  of  it  is  called  the  "  columella."  This 
asexual  form  of  multiplication  is  the  more  common,  but 
sexual  reproduction  occurs  under  conditions  not  well  defined. 


YEASTS    AND    MOULDS  335 

In  this  form  lateral  branches  (garnet ophores)  grow  out 
from  two  hyphae  close  to  each  other.  These  gametophores 
meet  by  the  tips,  fuse,  and  then  by  septa  the  central 
portion  is  separated  off  and  becomes  a  "  zygospore." 
The  mature  zygospore  under  suitable  conditions  enlarges 
and  sends  out  a  germ  tube  or  hypha,  on  the  end  of  which 
a  sporangium  may  appear.  Mucor  grows  on  gelatin  plate 
as  round  white  colonies  which  soon  cause  liquefaction.  In 
gelatin  stab,  it  forms  a  dense  white  growth  spreading  over 
the  surface,  and  sending  down  penetrating  branches — sub- 
aerial  hyphae  ;  others  rise  up  vertically  into  the  tube — 
aerial  hyphae.  Other  mucors  are  :  M.  stolonifer  (black 
mucor),  M.  spinosus  (chocolate  colour  :  has  spines  on  the 
columella) . 

Aspergillus  or  "Knob"  Mould. — This  is  a  common 
form  of  mould,  occurring  on  bread,  cheese,  oranges,  etc. 
There  are  several  varieties,  A.  glaucus  (blue  mould),  A. 
niger  (black  mould),  A.  flavus  (yellow  mould),  and  A. 
fumigatus  (green  turning  to  grey).  The  mycelial  filaments 
are  composed  of  numerous  rod-like  cells  joined  end  to  end. 
They  reproduce  asexually.  Hyphae  arise  from  the  mycelial 
network,  and  each  hypha  terminates  in  a  knob-like  expan- 
sion, the  columella.  The  surface  of  the  columella  becomes 
studded  with  flask-shaped  organs  or  cells  called  sterig- 
mata,  and  each  of  these  forms  spores  or  conidia,  which 
remain  attached  in  chains  like  streptococci.  The  result  is 
a  knob  with  radial  projections  composed  of  spores  ;  but 
having,  unlike  Mucor,  no  containing  capsule. 

Aspergilli  grow  on  gelatin  as  round  white  colonies  very 
like  those  of  penicillium.  In  a  few  days  coloured  points 
appear,  denoting  spore  formation,  being  blue  in  glaucus, 
black  in  niger,  etc.  The  gelatin  is  liquefied.  In  gelatin 
stab  there  is  a  dense  felt-like  growth  (more  pronounced 
than  with  penicillium),  and  later  liquefaction. 

The  pathogenic  aspergilli  include  : — 

i.  Aspergillus  fumigatus,  which  has  been  found  on  the 
one  hand  in  a  malady  simulating  pulmonary  tuberculosis, 
but  not  showing  tubercle  bacilli  in  the  sputum  (at  times 
it  is  associated  with  the  tubercle  bacillus)  ;  and  on  the 
other  hand,  causing  affections  in  the  external  auditory 
canal,  the  tympanic  cavity,  the  nasal  fossae,  and  in  wounds. 


336        PUBLIC    HEALTH    BACTERIOLOGY 

Such  infections  mostly  occur  in  those  engaged  in  handling 
grain,  whole  or  crushed,  either  as  transporters  or  as 
feeders  of  animals  or  fowls.  Many  of  the  cases  are  among 
bird  fanciers,  especially  those  keeping  pigeons,  which 
among  birds  are  specially  liable  to  aspergillosis.  Birds 
and  mammals  can  be  fatally  infected  by  intravenous 
inoculation  with  aspergillus  spores.  In  birds,  infection 
has  been  produced  by  inhalation  of  spores.  In  examining 
sputum  for  the  presence  of  aspergilli,  it  is  absolutely 
necessary  that  the  examination  should  be  made  immedi- 
ately after  expectoration,  since  the  spores  of  such  moulds 
may  exist  in  the  air  in  considerable  numbers,  and  falling 
on  to  the  sputum  would  germinate  there.  A  film  is 
made  on  a  slide  in  the  ordinary  way,  dried,  fixed  by  heat 
or  absolute  alcohol,  stained  with  carbol-thionin,  and 
examined.  The  characteristic  threads  or  filaments  of 
the  mycelium  are  seen  among  the  pus  cells.  It  is  further 
necessary  to  verify  the  diagnosis  by  cultivating  the  fungus 
and  noting  its  growth  and  morphology.  This  should  be 
done  on  a  special  medium,  such  as  Raulin's  liquid  medium, 
in  which  the  aspergillus  grows  well.  This  is  composed 
of  water,  1500  grm.  ;  crystallized  sugar,  70  grm.  ; 
tartaric  acid  and  ammonium  nitrate,  of  each  4  grm.  ; 
ammonium  phosphate  and  potassium  carbonate,  o-6  grm.  ; 
magnesium  carbonate,  0-4  grm.  ;  ammonium  sulphate, 
0-25  grm.  ;  sulphate  of  iron,  sulphate  of  zinc,  potassium 
silicate,  and  manganese  carbonate,  of  each  o-p7  grm. 
Take  a  sufficiency  in  an  Erlenmeyer  flask,  inoculate  by 
dropping  in  a  small  piece  of  the  sputum,  and  incubate  at 
370  C.  In  3  to  10  days  there  grows  a  whitish  meshwork, 
with  branches  bearing  spores,  green  at  first,  but  in  a  few 
days  becoming  smoke-black.  The  growth  is  examined 
microscopically,  and  its  characters  are  studied.  To  verify 
its  pathogenic  action,  an  emulsion  of  the  culture  is  injected 
into  the  ear- vein  of  a  rabbit.  The  animal  dies  in  several 
days  with  a  generalized  pseudo-tuberculosis. 

2.  Aspergillus   repens,    found    in    the    auditory   canal, 
producing  a  false  membrane. 

3.  Aspergillus  flavus,  in  chronic  ear  discharges. 
Penicillium,    or    "  Pencil "   Mould.— The    blue-green 

variety  of  this  form  of  mould,  Penicillium  glaucum,  is  the 


YEASTS    AND    MOULDS  337 

most  commonly  occurring  of  all  moulds.  In  this  genus, 
the  mycelial  threads  are  septate  or  many-celled.  Hyphse 
are  given  off,  and  from  the  end  of  each  hypha,  two  or 
more  short  pencil-like  branches  arise,  and  these  likewise 
give  origin  to  other  similar  branches.  These  last,  or 
further  set  of  branches,  produce  spores  or  conidia,  which, 
remaining  attached,  form  a  string  of  spores.  The  branches 
producing  the  spores  are  called  sterigmata,  and  the  inter- 
mediate branches  the  basidia  or  conidiophores.  The 
result  is  not  unlike  an  #-ray  photograph  of  the  arm,  in 
which  the  humerus  represents  the  hypha,  the  radius  and 
ulna — two  basidia  (omit  the  wrist),  the  metacarpus — five 
sterigmata  (say  three  from  the  radius  and  two  from  the 
ulna),  each  sterigma  bearing  spores — the  phalanges.  The 
spores  are  rounded  in  shape.  Penicillium  glaucum  grows 
on  bread  paste,  showing  at  first  a  white  fluffy  growth, 
becoming  either  green  or  blue,  as  the  spores  form.  It 
sometimes  is  covered  with  little  drops  of  dew-like  fluid. 

On  gelatin  plates  it  grows  as  small  round  colonies  of 
hair-like  filaments,  at  first  white  in  colour,  but  later 
greenish.  The  gelatin  is  liquefied.  In  gelatin  stab  a 
white  fluffy  layer  or  scum  rapidly  forms  on  the  top,  and 
descending  branches  run  into  the  gelatin,  as  well  as  hori- 
zontal ones  from  the  stab.  The  medium  becomes  bluish 
or  greenish,  and  liquefaction  takes  place.  Growths  on 
agar  and  potato  have  similar  characters. 

Penicillia  have  been  described  as  the  cause  of  chronic 
catarrh  of  the  Eustachian  tube,  and  of  gastric  hyper- 
acidity. 

Microsporon  furfur — Is  a  mould  first  described  in  1846, 
and  found  in  the  skin  affection  called  pityriasis  versicolor. 
It  is  composed  of  sinuous  hyphae,  3  micra  in  thickness, 
showing  right-angle  branches.  The  spores  are  large, 
3  to  5  micra  in  diameter,  and  are  formed  in  a  manner 
similar  to  that  in  penicillium. 

Pityriasis  versicolor  is  seen  in  persons  subject  to  profuse 
perspiration,  who  have  been  infected  with  the  spores  from 
the  air  or  elsewhere.  The  fungus  grows  in  the  superficial 
layers  of  the  epidermis,  forming  a  yellowish'  or  coffee- 
and-milk  coloured  patch,  usually  seen  on  the  chest  or 
abdomen  or  back.     Little  or  no  discomfort  is  caused  to 

22 


338         PUBLIC    HEALTH    BACTERIOLOGY 

the  person  affected.  The  diagnosis  is  easily  confirmed 
by  examining  a  scale  in  a  drop  of  liquor  potassas ;  or  the 
scale  may  be  teased  out  on  a  slide  in  a  drop  of  absolute 
alcohol,  and  then  stained  with  eosin.  The  filaments 
and  the  large  round  spores  are  readily  seen. 

Microsporon  minutissimum  is  a  mould  described  as 
the  cause  of  dhobie's  itch  or  erythrasma,  which  is  a  common 
affection  in  the  tropics.  It  is  mainly  seen  in  the  axillae, 
the  scrotal  region,  the  insides  of  the  thighs,  and  the 
submammary  folds.  Like  the  Microsporon  furfur,  it 
lives  a  simple  saprophytic  existence  in  the  epidermis, 
causing  reddish-brown  patches  with  an  abrupt  edge. 
When  a  scale  is  removed,  washed  with  ether,  teased  out 
in  acetic  acid,  allowed  to  dry,  washed  with  alcohol,  and 
stained  with  carbol-thionin,  the  fungi  can  be  seen  with 
the  microscope  as  slender  sinuous  filaments,  formed  of 
short  elements,  very  similar  to  bacilli,  from  destruction 
of  parts  of  the  filaments,  or  non-staining  of  these  parts. 

Sporotrichum  Beurmanni  is  a  mould  composed  of  a 
mycelium,  the  filaments  of  which  branch  in  all  directions. 
The  hyphae  are  i  to  2  micra  thick,  and  at  the  ends  of  these 
oval  spores  are  formed  (3  to  5  micra  by  1*5  to  3),  singly  or 
in  grape-like  clusters.  Spores  are  also  formed  around  the 
main  filaments,  or  apparently  so.  The  full  life-cycle  of  the 
sporotrichon  has  not  yet  been  worked  out,  and  its  exact 
classification  is  still  a  matter  of  doubt.  It  was  first  isolated 
by  Schenk  in  1898  from  refractory  subcutaneous  abscesses 
in  man.  De  Beurmann  and  Ramond  rediscovered  it  in 
granulomata  in  the  skin  in  1903.  Since  then  numerous 
cases  have  been  reported  in  France,  and  lately  two  cases 
have  been  reported  in  this  country  (Ofenhein,  Lancet, 
191 1,  Vol.  1,  page  659  ;  and  Norman  Walker  and  James 
Ritchie,  British  Medical  Journal,  1911,  Vol.  2,  pages  1-5. 
The  latter  article  is  accompanied  by  a  special  coloured 
plate  and  a  short  bibliography). 

Sporotrichosis  is  a  disease  characterized  by  cutaneous 
and  subcutaneous  tumours,  firm  and  indolent.  These  may 
ulcerate  and  discharge  a  viscid  homogeneous  pus  of  a 
yellowish-grey  colour.  The  tumours  have  been  in  the  past 
mistaken  for  those  due  to  syphilis  and  tuberculosis,  and  pot- 
assium iodide  and  tuberculin  injections  or  other  treatment 


YEASTS    AND    MOULDS  339 

administered.  When  iodide  was  given,  cure  was  often 
effected,  and  so  the  diagnosis  was  apparently  confirmed. 
The  ulcers  have  similarly  been  treated  as  syphilitic,  as 
lupus,  and  as  simple  pyogenic  ulcers.  In  many  cases 
there  is  a  history  of  a  minor  injury,  with  a  spread  from 
this  up  the  line  of  the  lymphatics,  with  tumour  formation 
and  breaking  down  at  various  points  en  route.  The 
lymphatic  glands  are  not  usually  enlarged,  the  fungus 
probably  not  reaching  them  at  an  early  stage.  The 
affection  is  usually  not  a  serious  one,  but  there  is  little 
tendency  towards  cure  if  left  untreated.  Stimulating 
local  treatment,  together  with  the  administration  of  large 
doses  of  potassium  iodide  (60  to  80  grains,  or  4  to  5  grm. 
daily)  is  quickly  followed  by  cure.  The  diagnosis  is  based 
on  the  direct  examinations  of  scrapings,  which  are  usually 
negative  except  for  spores,  oval  and  3  to  5  micra  long  ; 
and  by  cultivation.  Pus  from  an  unbroken  abscess  (if 
possible)  is  inoculated  freely,  since  the  parasitic  elements 
are  scarce,  on  broth,  glycerin  agar,  potato,  carrot,  etc., 
on  all  of  which  it  grows  well.  Growth  becomes  visible 
in  some  days,  and  gradually  increases.  On  agar,  the 
colonies  are  first  white  and  cream,  and  later  a  dirty  grey. 
On  carrot,  the  colour  is  first  yellow,  then  grey,  and  finally 
quite  black.  On  potato,  small,  white,  woolly  spots 
appear,  increase  in  size,  and  change  to  a  brownish  colour. 
Further  growth  results  in  heaped-up  masses  likened  to 
cerebral  convolutions.  "  In  gelatin  stab,  an  inverted 
fir-tree  growth  is  got,  but  no  liquefaction.  It  forms  acid 
specially  with  inulin  in  peptone  solution,  and  also  with 
glucose,  maltose,  galactose,  raffinose,  saccharose,  and 
mannite ;  but  with  lactose,  dulcite,  inosite,  adonite, 
sorbite,  and  salicin,  the  medium  remains  alkaline.  In 
no  case  was  there  any  gas  formation.  No  indol  formation 
was  observed.  The  organism  was  definitely  aerobic " 
(Ritchie,  loc.  cit.).  This  observer  has  also  studied  the 
organism  in  hanging-drop  agar  cultures,  and  found  that 
the  mycelium  formation  is  readily  noted  at  220  C,  but  at 
370  C.  few  filaments  are  formed ;  instead,  large  spores 
(5  micra)  from  which  short  stalks  sprouted,  each  bearing  a 
spore,  which  thus  formed  a  circle  round  the  central  body. 
The  latter  soon  degenerated.     This  suggests  the  reason 


340         PUBLIC     HEALTH     BACTERIOLOGY 

why  filaments  are  not  usually  found  in  the  pus  or  granulo- 
mata.  The  optimum  temperature  therefore  is  about 
20°  C.  (150  to  220).  The  organism  is  Gram-positive,  but 
not  acid-fast. 

Outside  the  human  body,  the  organism  has  been  found 
living  on  decaying  vegetable  matter.  Sporotrichosis  has 
also  been  described  as  occurring  in  dogs,  rats,  and  in  the 
horse.  In  the  last  it  causes  a  lymphangitis  with  superficial 
granulomata  of  a  benign  nature,  but  important  from 
having  a  resemblance  to  glanders,  especially  as  it  also 
at  times  prevails  as  an  epizootic.  Human  infection  from 
the  horse  has  been  reported  in  twelve  instances  in  seven  or 
eight  years  in  North  Dakota,  U.S.A.,  where  sporotrichosis 
in  horses  occurs  moderately  frequently.  A  case  is  also 
reported  in  a  female  bitten  on  both  thumbs  while  holding 
a  rat  which  had  been  inoculated  with  sporotrichosis. 
Serum  agglutination  and  complement  fixation  have  been 
found  to  occur  in  sporotrichosis,  but  are  not  specific,  as 
the  serum  of  patients  suffering  from  other  mycotic 
affections  (thrush,  actinomycosis)  reacts  to  Sporotrichum 
Beurmanni,  at  least  in  the  dilutions  tried. 

Oidium  albicans  (sometimes  called  Saccharomyces 
albicans)  is  the  cause  of  thrush  (Gr.  Soor ;  Fr.  Muguet), 
a  localized  disease  of  the  mouth  and  pharynx,  but  also 
at  times  attacking  the  oesophagus,  stomach,  small  intestine, 
caecum,  and  anus ;  besides  being  occasionally  found 
developing  on  the  vulva,  in  the  vagina,  on  the  pre- 
puce, and  the  glans  penis.  On  rare  occasions  it  has 
been  found  in  the  bladder,  the  kidney,  the  lungs,  the  brain, 
and  in  the  blood.  The  oidium  is  composed  of  cylindrical 
filaments,  made  up  of  joints  50  to  60  micra  long  by  3  to 
5  micra  in  diameter.  These  give  off  branches,  which  bear 
spores  by  constriction  at  their  free  ends.  Budding  is 
also  noted  when  grown  in  media  containing  sugars,  allying 
it  to  the  yeasts.  Two  varieties  are  described,  one  which 
liquefies  gelatin  and  produces  spores  ;  another  which  does 
not  liquefy  gelatin  and  yields  small  spores.  It  grows  only 
in  acid  media.  Like  the  yeasts,  it  can  ferment  sugars,  but 
is  not  so  powerful  as  they  are. 

It  is  easily  diagnosed  microscopically.  A  fragment  of 
the  white  membranous  growth  from  the  tongue  or  mouth 


YEASTS    AND    MOULDS  341 

is  taken  on  a  slide,  teased  out  in  a  drop  of  acetic  acid 
(which  renders  the  epithelial  cells  almost  invisible),  and 
examined.  The  parasite  is  clearly  seen  as  described 
above.  The  spores  are  round  or  oval.  A  stained  specimen 
may  be  made  by  teasing  in  a  distilled  water  drop,  exposing 
to  dry,  fixing  by  heat  or  absolute  alcohol,  and  staining 
with  thionin.  To  isolate  in  culture,  inoculate  on  gelatin, 
and  incubate  at  a  low  temperature  (150  to  200  C.)  for  48 
hours.  By  that  time  white  or  creamy  colonies  appear, 
which  are  pure  cultures  of  the  thrush  fungus.  The  ordinary 
microbes  of  the  mouth  are  unable  to  develop  at  that 
temperature  in  the  same  time. 

Ringworm  Fungi. — The  study  of  these  is  very  complex. 
The  mode  of  demonstration  of  them  in  the  various  parts 
affected  is  the  same  for  all,  and  is  summarized  thus  by 
Agasse-Lafont : — 

Hairs. — Prepare  a  solution  of  caustic  potash  of  30  per 
cent  strength.  Extract  some  of  the  diseased  hairs,  and 
put  them  in  a  drop  of  this  solution  on  a  slide.  Put 
on  a  cover-glass,  and  heat  moderately  for  several  seconds 
over  the  flame  of  a  spirit  lamp,  until  the  hairs  can  be 
crushed  by  gentle  pressure  on  the  cover-glass.  Examine 
directly  without  staining,  with  a  dry  lens  and  medium 
light  ;   or  first  mount  in  glycerin  or  glycerin  jelly. 

Epidermal  Scales. — Tease  out  with  two  sterile  needles, 
and  treat  in  the  same  manner. 

Nails. — Reduce  to  powder  with  a  nail  file,  and  treat  as 
above. 

Pus. — Dry  on  slide,  and  examine  directly  without 
staining. 

Favus  Crusts. — Tease  out,  crush  between  two  slides,  and 
thereafter  treat  as  for  hairs. 

They  are  best  cultivated  on  Sabouraud's  medium 
Agar,  18  grm.  ;  peptone,  10  grm.  ;  maltose,  40  grm  ;  and 
water  to  1000  c.c.  Heat  to  dissolve,  fill  into  tubes,  and 
sterilize  on  three  successive  days.  To  inoculate  tubes, 
take  an  infected  hair,  rinse  it  for  a  few  seconds  in  absolute 
alcohol,  and  wash  thoroughly  with  sterile  water.  Then 
stab  it  into  medium  at  several  places,  and  grow  at  180  C 
If  first  growth  is  not  pure,  remove  plug,  and  inverting  tube 


342 


PUBLIC    HEALTH    BACTERIOLOGY 


over  another,  tap  it  smartly,  when  spores  of  the  fungus 
will  fall  into  the  other  tube  and  inoculate  it. 

At  i8°  C.  growth  appears  in  seven  days  as  fine  white 
downy  tufts,  which  increase  in  size  and  throw  out  rays. 
The  surface  of  the  growth  becomes  covered  with  a  fine 
white  powdery  material.  If  grown  on  gelatin,  liquefaction 
takes  place  in  twelve  to  fifteen  days.  The  microsporon 
fungus  gives  a  more  delicate  growth  than  the  megalosporon 
fungus,  and  also  shows  microscopically  club-shaped  ends 
to  some  of  the  filaments,  which  are  not  found  in  the  other. 
In  both  forms,  spores  are  found  on  one  side  of  the  threads 
(like  the  teeth  of  a  crab)  or  at  ends  like  a  bunch  of  grapes. 

Table  of  the  Principal  Ringworm  Fungi  (Agasse-Lafont). 


Trichophyton 

Trichophyton 

Microsporon 

Achorion 

tonsurans 

mentagrophytes 

audouini 

schoenleinii 

Patho- 

Ringworm of 

Tinea  tonsur- 

Tinea ton- 

Favus of 

genic 

scalp :  Tinea 

ans  suppurates; 

surans,  etc. 

scalp,  skin, 

role 

tonsurans 

Body:  Herpes 

circinatus 

Tinea  barbce 

or 
Sycosis  menti 

and  nails 

Lesions 

Small : 

Small : 

Large : 

Sulphur- 

produced 

Hairs :  broken 

close  to  the 

scalp,  or  short 

Suppurative : 

Hairs;   bro- 
ken long, 
and  having 
at  their  base 
a  greyish 
sheath 

yellow  crusts 

Principal 

i.  Spores  large 

1.  Spores 

1.  Spores 

1.  Mycelia  of 

charac- 

w 

vary  (2-10^) 

small  (2~3M) 

varying  thick- 

.ters  of 

2.  In  "^regular 

2.  In  lines 

2. In  mosaics 

nesses 

fungus 

lines 

3.  Inside  and 

3.  Spores 

2.  Wavy 

3.  Inside  hair 

outside  hair 

only  on  out- 

roots 

roots 

side  of  roots 

Trichophyton  tonsurans. — This  fungus  is  the  cause  of 
30  per  cent  of  the  ringworm  of  the  scalp,  in  Paris,  and 
almost  all  the  cases  in  Germany  and  Italy.  In  the  east  end 
of  London,  among  the  Polish,  German,  and  Russian  Jews, 
it  is  a  common  cause.  It  also  is  found  in  ringworm  of  the 
body,  and  at  times  causes  an  affection  of  the  beard  and  the 
eyebrows,  dry  in  character.     Rarely,  it  affects  the  nails. 


YEASTS    AND    MOULDS  343 

It  is  composed  of  simple  filaments  interlaced.  In  the  hair 
bulbs,  the  filaments  are  inside  the  cuticle  (endothrix)  and- 
running  parallel  to  the  long  axis,  and  are  formed  of  cells  or 
spores,  almost  square.  These  spores  are  4  to  5  micra  long, 
and  are  regularly  arranged  in  lines.  It  is  readily  grown 
on  Sabouraud's  medium,  showing  in  five  to  six  days.  It 
liquefies  gelatin.  A  variety  is  quite  frequently  met  with 
in  the  same  places,  T.  Sabouraudi.  It  has  a  more  fragile 
mycelium,  and  shows  round  spores. 

Trichophyton  mentagrophytes  is  a  cause  of  sycosis 
menti,  a  suppurative  affection  of  the  beard,  and  also  of 
ringworm  of  the  body,  and  a  suppurating  ringworm  of  the 
scalp  in  infants.  The  filaments  are  composed  of  strings 
of  round  spores  of  varying  diameters  (2  to  10  micra).  The 
filamentary  threads  are  mostly  outside  the  hair  cuticle  ; 
(endo-  and  ectothrix)  ;  a  few  are  found  inside  but  towards 
the  periphery. 

Microsporon  or  Microsporon  Audouini  is  almost  the 
sole  cause  of  ringworm  of  the  scalp  in  Scotland,  and  of 
96  per  cent  of  the  cases  in  London  among  British  subjects. 
It  is  called  the  small-spored  fungus  as  compared  with  the 
two  given  above,  which  are  called  "  megalosporon,"  or 
large-spored.  The  parasite  encloses  the  diseased  hair  in  a 
whitish  case  formed  of  a  mosaic  of  spores  (ectothrix) .  The 
spores  are  2  to  3  micra  in  diameter,  and  from  pressure 
against  one  another  in  the  mosaic  pattern,  become  poly- 
hedral in  shape.  When  stained  with  carbol-thionin,  the 
filaments  are  seen  in  the  interior  of  the  hair. 

Achorion  Schoenleinii  is  the  fungus  which  causes  f avus, 
or  honeycomb  ringworm,  in  which  the  characteristic 
feature  is  the  formation  of  cup-shaped  crusts  of  a  sulphur- 
yellow  colour.  It  most  commonly  attacks  the  scalp,  but 
also  affects  the  skin  of  the  body  and  the  nails.  When 
attacking  the  nails,  they  become  yellow  and  thickened. 
Besides  the  characteristic  form  of  attack,  the  fungus  may 
also  produce  a  moist  dermatitis  resembling  that  due  to 
the  other  ringworm  fungi. 

In  the  hair  the  parasite  is  seen  as  wavy  lines  of  mycelia 
composed  of  spores.  The  spores  are  irregular  in  size  and 
shape,  but  mostly  polyhedral.  Rarely,  the  mycelium  is 
septate  and  without  spores. 


344         PUBLIC     HEALTH    BACTERIOLOGY 

Favus  prevails  also  in  mice  and  cats,  and  from  the 
latter  animals  many  of  the  human  cases  arise. 

The  Achorion  Schoenleinii  in  culture  grows  best  at 
300  to  350  C,  and  scarcely  grows  at  io°  to  150  C,  unlike  the 
other  ringworm  fungi.  It  also  liquefies  gelatin  more 
quickly  than  they,  namely,  in  three  to  four  days.  It 
forms  snowy-white  circular  or  oval  colonies,  becoming 
finely  powdered  over  the  surface,  and  wrinkled  in  old 
culture.  It  grows  best  on  beer-wort  agar.  To  beer- wort 
diluted  to  a  specific  gravity  of  1100  add  1-5  per  cent 
of  agar.  Heat  for  two  hours  until  dissolved.  Filter, 
tube,  and  sterilize.     (Avoid  overheating.) 


CHAPTER    XV III. 

SPECIAL 

BACTERIOLOGICAL     EXAMINATIONS. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER. 
In  all  natural  unfiltered  waters,  except  when  derived 
from  deep  wells  and  springs  (in  which  case  filtration  has 
already  taken  place  through  the  strata)  numbers  of  bacteria 
are  found.  The  actual  content  is  determined  by  the 
accumulated  action  of  the  following  factors,  namely  : — 

i.  Presence  or  absence  of  local  pollution. 

2.  Presence  or  absence  of  natural  purification. 

3.  The  season  of  the  year. 

4.  The  rainfall  at  any  particular  period. 

The  bacteria  found  in  water  may  likewise  be  classed 
under  four  heads,  as — 

1.  Harmless  :  the  natural  water  bacteria.  Such  are  the 
B.  fluorescens  liquefaciens,  B.  fluorescens  non-liquefaciens, 
B.  prodigiosus,  B.  violaceus,  sarcinae,  and  spirilla.  These 
all  grow  best  at  room  temperature. 

2.  Unobjectionable  :  those  present  from  soil  washings, 
as  B.  subtilis,  B.  mycoides,  and  B.  megatherium. 

3.  Objectionable :  those  derived  from  sewage,  either 
directly  or  from  sewage-polluted  soil.  Such  are  :  (a)  The 
B.  proteus  group  ;  (b)  B.  coli  communis  and  its  allies ; 
(c)  Streptococci  ;  (d)  Staphylococci  ;  (e)  B.  enteritidis 
sporogenes. 

4.  Dangerous  :  those  capable  of  causing  infection  by  the 
alimentary  canal.  Such  are  the  B.  typhosus,  B.  para- 
typhosus,  B.  dysenteriae,  and  Sp.  cholerae. 

Samples. — Stoppered  sterile  glass  bottles  should  be 
used  for  sampling,  each  of  at  least  250  ex.  capacity  (8  to 
10  oz.).  The  bottle  should  be  thoroughly  cleansed  with 
soap  and  water,  well  rinsed  with  clean  water,  and  sterilized 
(inverted  and  with  stopper  out)  in  steamer  for  one  hour. 


346 


PUBLIC    HEALTH    BACTERIOLOGY 


Allow  the  sterilizer  to  cool,  then  stopper  the  bottle,  and 
remove  and  put  in  case. 

In  sampling  from  a  tap,  run  the  water  to  waste  for  half 
an  hour,  and  then  fill  bottle.  Stopper,  and  label  with 
particulars  of  place,  time,  and  date.  Examine  at  once, 
or  pack  in  ice  to  prevent  multiplication  of  organisms. 

In  sampling  from  a  lake,  dip  stoppered  bottle  well  below 
surface  ;  remove  stopper,  and  keep  it  under  water  ;  allow 
bottle  to  fill ;  replace  stopper,  and  bring  bottle  to  surface. 
Pack  in  ice. 

Examine  samples  as  soon  as  possible  after  collection. 
Keep  in  ice  in  meantime. 

Dilutions. — If  the  water  is  pure,  no  dilution  will  be 
required  ;  if  impure,  varying  dilutions  are  used  according 
to  the  degree  of  impurity.  These  may  be  made  by  the 
decimal  mode  of  dilution  described  on  page  367  ;  or  flasks 
may  be  kept  ready  containing  100  c.c.  of  sterile  water. 
One  c.c.  of  sample  added  to  such  a  flask  by  sterile  pipette 
gives  practically  a  dilution  of  1  in  100  ;  1  c.c.  from  this 
flask  to  another  sterile  100  c.c.  gives  1  in  10,000  ;  and  so  on. 
To  get  1  in  10,  remove  10  c.c.  by  sterile  pipette,  and  add 
10  c.c.  of  sample  ;  and  from  this  dilution  others  are  simi- 
larly made. 

Standards. 


Deep  wells  and  springs   \ 

Surface  waters  : — 
Shallow  wells 
Cultivated  lands 
Rivers 


Bacterial  Count. 


Gelatin  Plate 
at  200  C. 


Should  not 

exceed 
50  per  c.c. 

Ditto 
500  per  c.c. 


Agar  Plate 
at  37°  C. 


Should  not 

exceed 
10  per  c.c. 

Ditto 
50  per  c.c. 


Bacillus 

Coli 

Communis. 


Should  be 

absent 
in  100  c.c. 

Ditto 
in  10  c.c. 


Methods  of  Water  Examination. — These  are  based 
on  the  knowledge  that  the  dangerous  organisms  in  water 
are  usually  present  from  sewage  pollution.  Inasmuch  as 
some  of  these  forms  are  not  easily  isolated  from  water,  the 
mode  of  procedure  is  to  ^enumerate  the  total  bacterial 
content,  and  to  look  for  an  organism,  likewise  present  from 


SPECIAL    EXAMINATIONS  347 

sewage  pollution,  but  easily  found  if  present.  Such  an 
organism  is  Bacillus  coli  communis,  which  is  present  in 
enormous  numbers  in  the  sewage  of  man  and  animals  ; 
and  is  therefore  likely  to  be  present  in  sewage  contamin- 
ated water,  even  after  great  dilution.  The  B.  coli  is 
also  a  more  resistant  organism  than  the  dangerous  forms, 
and  so  serves  to  indicate  pollution  at  a  later  stage  than 
these  could  possibly  be  found  in  a  water.  So  far  it  is  not 
possible  to  distinguish  B.  coli  of  human  origin  from  those 
of  animal  origin.  It  is  stated  that  those  of  human  origin 
are  more  pathogenic  to  animals. 

There  are  various  methods  in  use  in  this  country.  A 
Committee  of  the  Royal  Institute  of  Public  Health 
appointed  to  consider  the  "  Bacterioscopic  Examination  of 
Water,"  reported  in  1904  {Journal  of  State  Medicine,  vol. 
xii,  p.  471)  as  follows  : — 

Minimal  Procedure.     Unanimous  report : — 

(a.)  Enumeration  of  bacteria  present  in  a  water 
sample,  capable  of  growing  on  a  medium  incubated 
at  room  temperature  (i8°-22°  C). 

(b.)  Search  for  Bacillus   coli,   and  identification 
and  enumeration  of  this  organism,  if  present. 
Majority  also  recommended  : — 

(c.)  Enumeration  of  bacteria  present  in  sample 
capable  of  growing  on  a  medium  incubated  at  blood 
heat  (360-38°  C). 

(d.)  Search  for  and  enumeration  of  streptococci. 
(e.)  Do  not  recommend  routine  examination  for 
Bacillus  enteritidis  sporogenes ;    but  in  exceptional 
and  special  cases  advise  that  it  be  searched  for. 

They  further  report  on  mode  of  collection  of  sample, 
media  to  be  used  in  the  tests,  etc.,  of  which  the  following 
is  a  brief  summary  : — 

Collection  of  Sample. — The  sample  should  be  collected  in 
sterile  stoppered  glass  bottles  (minimum  quantity  60  c.c,  or 
2  ounces),  and  should  be  packed  in  ice.  At  least  10  ounces 
of  sample  should  be  sent,  and  its  examination  should  be  begun 
within  three  hours,  or  it  should  be  left  packed  in  ice. 

Enumeration  of  Bacteria. — The  media  to  be  used  are  all  to 
be  standardized  to  have  a  reaction  of  +10  (Eyre's  scale). 
Owing  to  changes  in  reaction,  media  should  not  be  more  than 


348         PUBLIC    HEALTH    BACTERIOLOGY 

three  weeks  old.  For  cultivation  at  room  temperature  a 
choice  may  be  made  from  the  following :  Distilled-water 
gelatin,  nutrient  gelatin,  distilled-water  agar,  nutrient  agar, 
and  gelatin  agar.  At  blood  heat  use  agar  or  gelatin  agar. 
Both  agar  and  gelatin  media  should  be  used  in  any  one  test. 
Polluted  water  gives  more  colonies  on  nutrient  gelatin  than 
on  distilled-water  gelatin  ;  unpolluted  water  gives  more  with 
distilled-water  gelatin. 

The  size  of  the  plates,  the  amount  of  medium  to  be  used  in 
plating,  and  the  amounts  of  sample  to  be  added  to  the  media 
are  all  specified.  Plates  should  be  10  cm.  in  diameter  ;  10  c.c. 
of  medium  should  be  used  ;  and  for  ordinary  waters,  o«2  c.c, 
0-3  c.c,  and  0-5  c.c.  of  sample  should  be  added  to  gelatin 
media,  and  o-i  c.c.  and  i*o  c.c  of  sample  to  agar  media.  The 
sample  should  be  thoroughly  shaken  before  removing  these 
amounts,  and  the  tubes  should  be  thoroughly  mixed  by  rota- 
tion before  plating.  Duplicates  should  be  put  up.  In  an 
unknown  water,  additional  plates  of  dilutions  (ten  and  one 
hundred  fold)  should  be  made.  The  colonies  should  be  counted 
by  the  naked  eye,  and  preferably  by  daylight.  A  lens  may  be 
used  for  doubtful  colonies.     The  time  of  counting  should  be  : 

For  gelatin  plates,  at  the  end  of  72  hours  (3  days)  ; 

For  agar  plates,  after  40  to  48  hours. 

The  gelatin  plates  should  be  inspected  daily,  in  case  counting 
becomes  necessary  earlier  from  liquefaction  of  the  medium. 

Search  for  B.  Coli. — MacConkey's  broth  is  recommended 
to  be  used,  the  sample  to  be  added  directly  to  the  medium, 
and  not  first  concentrated  by  filtration. 

Isolation  of  B.  Coli. — If  indications  of  the  presence  of 
B.  coli  are  got,  then  the  organism  must  be  isolated,  cultivated, 
and  identified.  This  is  advised  to  be  done  by  making  surface 
cultures  on  plates  of  either  : — 

(a.)  Litmus  lactose  agar  (reaction  +10)  ; 
or  (b.)  Bile-salt  lactose  agar  (MacConkey's)  ; 
or  (c.)   Nutrose  lactose  agar  (Drigalski  and  Conradi)  ; 
or  (d.)  Ordinary  nutrient  gelatin. 

They  consider  (c)  to  be  the  best  medium  of  all.  Agar  media 
save  time. 

Identification  and  Tests. — Having  obtained  coli-like  colonies 
on  plates  made  from  the  preliminary  cultivations  of  the  water 
in  MacConkey's  broth,  subcultures  must  be  made  to  identify 
the  organism.  The  following  subcultures  should  at  least  be 
made  : — 

(a.)  Surface  Agar,  on  which  the  abundant  growth 
enables  subcultures,  etc.,  to  be  easily  made  if 
required. 


SPECIAL    EXAMINATIONS  349 

{b.)  Stab  and  surface  cultures  in   gelatin.     These  may- 
be done  in  the  same  tube. 
(c.)   Litmus  milk  incubated  at  37  °  C. 
(d.)  Glucose  litmus  medium. 
(e.)   Lactose  litmus  medium. 
(/.)    Peptone  water  for  indol  reaction. 

The  Committee  consider  an  organism  to  be  typical  B.  coli 
when  it  conforms  to  the  following  characteristics :  Small 
motile  bacillus,  non-sporing,  decolorized  by  Gram,  which 
grows  well  at  37  °  C.  and  at  room  temperature  ;  never  liquefies 
gelatin  ;  produces  permanent  acidity  in  milk  ;  curdles  milk 
within  seven  days  at  370  C.  ;  ferments  glucose  and  lactose, 
with  formation  of  acid  and  gas  ;  grows  in  smooth  thin  surface 
on  gelatin  (not  corrugated)  ;  and  in  gelatin  stab  grows  well 
to  the  bottom  of  stab.  This  typical  B.  coli  generally  also 
forms  indol,  gives  a  thick  yellowish- brown  growth  on  potato, 
changes  neutral-red,  reduces  nitrates  to  nitrites,  and  in  fer- 
menting glucose  half  of  the  gas  produced  is  absorbed  by  KOH. 
It  sometimes  ferments  saccharose. 

The  method  here  described  is  that  used  in  the  City 
Bacteriological  Laboratory,   Glasgow. 

I.  Enumeration  of  Bacteria. — Add  o-i  c.c.  and  1  c.c. 
of  sample  by  sterile  pipette  to  10  c.c.  of  liquefied  gelatin 
and  agar.  Mix  thoroughly  by  rotation  and  plate.  Incubate 
the  gelatin  plates  at  20 °  C.  ;  inspect  daily,  and  count  after 
three  days,  unless  necessary  earlier. 

Incubate  the  agar  plates  at  370  C,  and  count  after  two 
days. 

The  ordinary  water  bacteria  grow  best  on  gelatin, 
whereas  the  intestinal  forms  grow  best  on  the  agar  at 
blood  heat.  Hence,  in  a  pure  water  the  gelatin  count 
should  be  much  the  greater ;  and  in  an  impure  water  the 
difference  between  the  counts  becomes  less  marked  the 
more  impure  the  water.  The  ratio  between  the  two 
counts  is  also  noted.  This  ratio  of  the  number  of  organisms 
developing  at  room  temperature  to  the  number  at  blood- 
heat  =  10  :  1  in  pure  water  and  =  10  :  2  or  3  or  5,  etc.,  in 
polluted  waters.  The  ratio  is  unreliable  in  surface  waters 
in  tropical  countries,  because  B.  liquefaciens,  B.  fluorescens 
liquefaciens,  and  B.  fluorescens  non-liquefaciens  are  com- 
monly present,  and  all  grow  well  at  37°C.,  and  are  harmless. 


350 


PUBLIC    HEALTH    BACTERIOLOGY 


Average  Number  of  Bacteria  per 
Growing  on  Gelatin 

c.c.  of  Water  Sample 

AT    20°    C. 

Glasgow  Tap  Water. 

Raw  Thames  Water. 

Year  1909  : — 
Highest 

Lowest 
Average  j 

73    (Dec.).. 

31   (June).. 
54*2 

19,794   (Dec.)   river  in 
flood. 
913  (May)  river  low. 
3,818. 

Year  1910  : — 
Highest 

Lowest 
Average 

105      (Jan.)  .  . 

19-5  (May)  .  . 
43-o 

22,939   (Dec.)   river  in 
flood. 
1,522  (May)  river  low 
7>4i°- 

Dr.  A.  C.  Houston  (Director  of  Water  Examination  to 
the  Metropolitan  Water  Board,  London),  in  addition 
to  gelatin  and  agar  media,  for  enumeration,  also  uses 
MacConkey's  neutral-ral,  fo'le-salt,  peptone,  lactose  3  gar 
(called  rebipelagar  for  brevity).  This  is  similarly  inocu- 
lated and  plated  and  incubated  at  37 °.  The  colonies  are 
counted  on  gelatin  on  the  third  day,  and  on  agar  and  rebi- 
pelagar after  twenty  to  twenty-four  hours. 

The  following  table,  culled  from  Dr.  Houston's  reports, 
serves  to  illustrate  the  ratios  of  the  various  counts  : — 


RAW    RIVER    THAMES    WATER. 

Average  Number  of  Microbes  per  c.c.  in  Comparable  Samples, 

Tested  on  Three  Media  ;  with  Ratios. 


Gelatin. 

Agar. 

Rebipelagar. 

Ratio. 

Ratio. 

Year. 

Gelatin. 
Agar. 

Agar. 

Rebipelagar. 

1908-09 

2745 

319 

38 

8:  I 

8:  1 

I909-IO 

531° 

495 

63 

11  :  1 

8:  1 

1910-II 

6184 

339 

20 

18:  1 

17:  1 

Gelatin  at  2o°-22°  C.  ;   colonies  counted  on  third  day. 
Agar  and  Rebipelagar  at  370  C.  ;  counted  after  twenty  to  twenty- 
four  hours. 

This   further   table    (curtailed)    from    Houston's   Fifth 


SPECIAL    EXAMINATIONS 


351 


Annual  Report  (page  7)  shows  the  influence  of  rainfall  in 
the  Thames  valley  on  the  average  daily  flow  of  the  River 
Thames,  and  on  the  bacterial  content  of  the  raw  river 
water  : — 


Average 

daily 

(natural)  flow 

of  the  River 

Thames  in 

million 

gallons. 

Total  number 

Percentage 

number  of 
samples  of  raw 
Thames  Water 

containing 
typical  B.  Coli 

in  o*i  c.c, 

Raw  Thames 

Month. 

Rainfall 
(inches) 
Thames 

Valley. 

of  bacteria 

per  cc.  in  the 

raw  Thames 

Water 

(gelatin) 

Water.  Oxygen 
absorbed  from 
permanganate 

test  (parts 
per  100,000) 

1910  : 

April 

2-13 

1353 

3IO9 

23'7 

•1352 

May 

I'96 

979 

1522 

12-5 

•I489 

June 

3-04 

1149 

2721 

500 

•3031 

July 

2-25 

795 

2589 

526 

•1756 

August 

2-88 

589 

2702 

13*7 

•1357 

September 

0*46 

501 

3035 

13-6 

•1173 

October    .  . 

3-48 

666 

3736 

38-1 

•1611 

November 

361 

1595 

17932 

682 

•3249 

December 

5-21 

5064 

22939 

83-3 

•5253 

1911  : 

January    .  . 

1*21 

2657 

10438 

857 

•1852 

February 

1-67 

1443 

8035 

700 

•1140 

March 

1-99 

2033 

9300 

78-2 

•2495 

Sum 

3°'49 

Averages 

2-54 

1574 

7324 

49-4 

•2154 

Note. — The  figures  in  bold  type  exceed  their  respective  averages. 

II.  Search  for  B.  Coli. — For  this  purpose  MacConkey's 
broth  is  used  of  the  composition  neutral-red,  bile-salt, 
peptone,  glucose  water,*  in  single,  double,  triple  and  quad- 
ruple strengths.  The  proper  quantities  are  put  into 
suitable  sized  tubes,  and  fermentation  or  Durham's  tubes 


*  Just  as  neutral-red,  bile-salt  peptone,  lactose,  agar  has  been 
shortened  to  rebipelagar  (see  p.  350),  so  MacConkey's  broth  with 
the  various  carbohydrates  might  be  written  thus  : — ■ 

Neutral-red,  bile-salt,  peptone,  glucose  water  (aqua)  as  rebipegluqua. 

lactose  „         „       rebipelaqua. 


saccharose 

„         „       rebipesaqua. 

dulcite 

„         „       rebipeduqua 

mannite 

,         „       rebipemaqua. 

adonite 

,         „       rebipeadqua. 

inulin              , 

,         „       rebipeinqua. 

352  PUBLIC    HEALTH    BACTERIOLOGY 

added,  and  the  whole  sterilized.  Thereafter,  the  sample, 
having  been  well  shaken,  is  added  direct  by  sterile  pipette, 
and  always  without  concentration  by  filtration.  If  the 
sample  is  too  strong,  suitable  dilutions  are  made,  and  i 
c.c.  of  the  dilution  is  added.  In  the  case  of  an  unknown 
water  the  following  tubes  would  be  put  up  : — 

o-oooi  c.c.  of  sample  to  10  c.c.  of  single  strength  medium. 


o-ooi 

J  J                        J) 

o-oi 

>>                      ) 

o-i 

>>                       y 

i-o 

,,                       , 

io-o 

>>                       > 

50*0 

>>                       > 

ioo-o 

)  >                       > 

of  double  strength. 
25   c.c.  of  triple  ,, 

30  c.c.  of  quadruple   ,, 

The  tubes  are  put  in  a  nest  or  basket,  and  incubated  at 
370  C.  for  twenty-four  hours.  The  possible  results  are 
four  : — 

(a.)  Acid  and  gas. 

(b.)  Acid,  no  gas. 

(c.)  No  acid,  no  gas  ;  turbidity. 

(d.)  No  visible  change. 

Interest  lies  in  (a)  and  (b),  and  they  are  commonly 
associated  ;  but  the  absence  of  (a)  from  all  the  tubes 
should  not  be  held  as  precluding  the  necessity  for  further 
investigation.  Note  the  tubes  showing  acid  and  gas,  or 
acid  alone  ;  say  that  the  tube  containing  least  amount  of 
sample,  and  which  shows  acid  and  gas,  is  that  to  which 
o- 1  c.c.  of  sample  was  added,  then  the  result  is  stated  thus  : 
Sample  showed  acid  and  gas  formation  down  to  o-i  c.c. 
As  a  rule  all  the  higher  tubes  will  show  acid  and  gas  too. 

Some  workers  call  this  the  "  presumptive  B.  coli  test ;  '* 
but  as  the  following  paragraphs  (from  Notter  and  Firth) 
show,  the  test  is  only  a  step  on  the  way  towards  the  isolation 
of  B.  coli. 

MacConkey's  Neutral-red,  Bile-salt,  Peptone,  Glucose  Water. 
Reaction  of  certain  bacteria  with  : 

Group  i.  Bacteria  producing  acid  -f-  gas. 

B.  coli  communis,   B.   enteritidis   (Gaertrier),  B. 
paracolon,  B.  paratyphosus,  B.  pneumoniae,  B.  lactis 


SPECIAL    EXAMINATIONS  353 

aerogenes,  B.  acidi  lactici,  B.  neapolitanus,  B.  icter- 
oides,  B.  psittacosis,  B.  cloacae,  B.  proteus  vulgaris, 
bacillus  of  hog  cholera,  bacillus  of  epidemic  jaundice, 
B.  oxytocus  perniciosus,  and  B.  capsulatus. 

Group  2.    Bacteria  producing  acid,  but  not  gas. 

B.  typhosus,  B.  dysenteriae,  B.  cholerae,  B.  pyo- 
genes fcetidus,  streptococci  and  staphylococci. 

The  first  group  can  be  subdivided  into  four  : — 

(a.)  The  proteus  group  of  motile  bacilli :  gelatin-lique- 
fying, form  acid  and  gas  in  glucose,  maltose,  saccharose, 
and  galactose,  but  not  in  lactose,  laevulose,  arabinose, 
rafhnose,  mannite,  sorbite,  dulcite,  adonite,  dextrin,  starch, 
or  inulin ;  curdle  milk  slowly  with  acid ;  commonly  produce 
indol  in  peptone  solutions. 

(b.)  B.  coli  communis  family  :  motile  bacilli,  non-gelatin- 
liquefying,  producing  acid  and  gas  in  all  the  above  except 
saccharose,  adonite,  starch,  or  inulin ;  curdle  milk  rapidly 
with  acid,  but  do  not  peptonize  clot ;   form  indol. 

(c.)  B.  lactis  aerogenes  group  :  non-motile  bacilli,  non- 
gelatin-liquefying,  producing  acid  and  gas  in  all  the  above 
except  three — dulcite,  adonite,  and  inulin ;  curdle  milk 
rapidly  with  acid  ;  do  not  peptonize  clot  ;   form  indol. 

(d.)  The  paracolon-enteritidis  series  :  motile  bacilli,  non- 
gelatin-liquefying,  producing  acid  and  gas  in  all  but  lactose, 
saccharose,  adonite,  starch,  or  inulin  ;  do  not  clot  milk 
but  finally  render  it  alkaline  ;   do  not  form  indol. 

III.   Isolation  of  B.  Coli.— 

(a.)  From  each  tube  showing  acid  and  gas,  and  acid 
alone,  make  a  surface  smear  with  one  platinum  loopful  on 
a  plate  of  neutral  red,  bile  salt,  peptone,  lactose,  agar  (rebi- 
pelagar)  containing  1  part  of  crystal  violet  in  10,000. 
Incubate  these  plates  for  twenty-four  hours  at  370  C. 

(b.)  Examine  thereafter.  If  a  plate  shows  only  one  kind 
of  colony,  inoculate  an  agar  slope  from  a  mixture  of  these. 
If  more  than  one  form  of  colony  is  seen  on  any  plate, 
inoculate  agar  slopes  from  each  kind  of  colony.  Incubate 
the  various  inoculated  agar  tubes  (properly  marked  or 
labelled)  at  370  C.  for  twenty-four  hours.  This  growth  on 
agar  gives  sufficient  material  for  subsequent  steps  ;  but 
it  is   also   necessary   to   revive  any  fermentative   powers 

23 


354 


PUBLIC    HEALTH    BACTERIOLOGY 


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SPECIAL    EXAMINATIONS 


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356         PUBLIC    HEALTH    BACTERIOLOGY 

possessed  by  any  of  the  bacteria,  as  these  are  found  to 
be  depreciated  by  growth  on  the  plate  medium. 

(c.)  From  each  growth  on  agar  the  following  subcultures 
are  made  :  Gelatin  stab,  litmus  milk,  peptone  water,  and 
seven  tubes  of  MacConkey's  broth,  containing  respec- 
tively the  following  carbohydrates :  Glucose,  lactose, 
saccharose,  dulcite,  mannite,  adonite,  inulin. 

The  gelatin  is  incubated  at  20°  C.  for  eight  to  ten  days. 
The  other  tubes  are  incubated  at  370  C.  for  seven  days. 
An  organism  which  does  not  liquefy  gelatin,  and  which 
gives  acid  and  clot  in  litmus  milk,  indol  in  peptone  water, 
and  acid  and  gas  in  glucose,  lactose,  dulcite  ,and  mannite, 
but  not  in  saccharose,  adonite,  or  inulin,  is  called  Bacillus 
coli  communis.  The  nature  of  organisms  giving  varying 
reactions  from  these  may  be  established  from  the  preced- 
ing table,  for  which  I  am  indebted  to  Dr.  R.  M.  Buchanan. 
Where  a  reaction  here  given  contradicts  that  stated  else- 
where in  the  text,  it  serves  to  show  that  different  results 
have  been  observed. 

Houston  adopts  a  different  method  of  arriving  at  what 
he  calls  a  "  typical  B.  coli  "  characteristic  of  excremental 
pollution. 

He  likewise  uses  MacConkey's  glucose  broth  (rebipegluqua), 
but  incubates  at  37 °  C.  for  two  days  (forty-eight  hours).  If 
no  gas  developed,  the  result  was  considered  negative.  On 
the  other  hand,  if  gas  developed,  he  then  made  subcultures 
from  tubes  showing  acid  and  gas  on  to  gelatin  slopes,  and 
incubated  for  two  days  at  20°-22°  C.  If  no  colonies  developed 
resembling  in  any  way  that  of  B.  coli,  the  result  was  considered 
negative.  If  coli-like  colonies  were  present,  one  of  the  most 
typical  looking  colonies  was  chosen  for  subculture  into  glucose 
gelatin,  and  a  "  shake  "  culture  made  in  the  customary  way. 
After  twenty-four  hours'  incubation  at  2o°-22°  C,  if  no  gas 
developed,  the  result  was  entered  as  negative  ;  just  as  if  no 
growth  had  occurred  in  the  oblique  gelatin  cultures,  or  a 
growth  of  microbes  in  no  way  resembling  B.  coli. 

But  if  gas  production  was  noted,  the  result  was  recorded 
as  positive,  and  the  other  biological  attributes  of  the  coli-like 
microbe  were  studied  in  neutral-red  broth  cultures  for 
fluorescence  (fl)  ;  in  lactose  peptone  cultures  for  acid  and  gas 
formation  (ag)  ;  in  peptone  water  cultures  for  indol  formation 
(in)  ;  and  in  litmus  milk  cultures  for  acid  clotting  of  the 
medium  (ac).     The  complete  combination  of  these  positive 


SPECIAL    EXAMINATIONS  857 

characters  was  expressed  by  the  word  "  flaginac."  Since 
January,  1907,  he  has  modified  his  method  in  this  wise. 
Finding  that,  practically  speaking,  a  "flaginac"  B.  coli  is  a 
B.  coli  indistinguishable,  according  to  the  tests  used,  from  the 
typical  B.  coli  of  excremental  pollutions  ;  and  that  in  the 
great  majority  of  cases  a  glucose  fermenting  coli-like  microbe 
will  also  produce  fluorescence  (fl)  in  neutral-red  broth  cultures  ; 
and,  further,  that  lactose  fermenting  (ag)  coli-like  microbes 
nearly  always  clot  milk  (ac)  ; — there  is,  therefore,  justification 
for  omitting  the  neutral-red  broth  and  litmus  milk  tests,  and 
relying  solely  on  the  lactose  peptone  (ag)  and  the  indol  (in) 
tests.  Practically,  in  the  case  of  the  London  waters,  an  "agin  " 
B.  coli  may  be  regarded  as  presumably  a  "  flaginac  "  B.  coli 
(Report  for  January,  1907).  On  this  basis  he  has  modified 
the  above  method,  in  order  to  make  it  more  rapid.  The  first 
growth  is  as  before,  on  MacConkey's  glucose  broth  for  forty- 
eight  hours.  The  secondary  cultures  are  on  rebipelagar 
(instead  of  gelatin)  for  twenty-four  hours.  From  red  coli-like 
colonies  on  this  medium,  subcultures  are  made  on  glucose, 
lactose,  and  saccharose,  gelatin  media  and  in  peptone  water. 
After  twenty-four  hours  gas  production  in  the  carbohydrate 
media  is  considered  positive  ;  and  indol  is  tested  for  in  the 
peptone  water.  Fermentation  of  lactose  and  production  of 
indol  are  looked  for,  "  agin  "  results  ;  if  saccharose  is  also 
fermented,  the  result  is  recorded  asa"  sagin  "  one.  (For  full 
description  see  the  January,  1907,  Report.)  In  this  way 
Houston  claims  that  "  it  becomes  possible  to  complete  the 
tests  for  excremental  pollution  in  water  within  four  days." 
It  will  be  noticed  that  Houston  accepts  asa"  type  of  B.  coli  " 
an  organism  fermenting  saccharose.  His  statement  on  the 
subject  is  as  follows  : — 

"  The  typical  B.  coli  of  the  human  intestinal  tract  may  be 
divided  into  two  classes,  according  to  their  action  on 
saccharose.  The  majority  do  not  ferment  this  sugar,' or  "act 
on  it  only  to  a  slight  extent.  Hence  typical  B.  coli  which 
do  not  ferment  saccharose  would  seem  to  be  more  significant 
of  undesirable  pollution  than  those  which  do  ferment  it." 

The  student  should  note  that  Houston  uses  the  term 
"  typical  B.  coli  "  on  a  different  basis  from  many  other 
bacteriologists,  who  would  be  inclined  to  speak  of  his  "  typical 
B.  coli  "  rather  asa"  coliform  organism." 

Houston  has  found  that  out  of  every  100  coliform  organisms 
present  in  raw  Thames  water,  70  to  80  per  cent  conform  to 
his  "  typical  B.  coli  "  ;  whereas  the  same  water  after  storage 
and  filtration  shows  a  reduction  in  the  coliform  organisms 


358         PUBLIC    HEALTH    BACTERIOLOGY 

which  is  more  marked  among  the  "  typicals,"  which  now 
number  40  to  50  per  cent  only  of  the  total  coliforms.  "  This 
means  that  the  ratio  or  proportion  between  the  typical  B.  coli 
and  the  B.  coli  (both  typical  and  non-typical)  is  altered  during 
the  purification  processes  in  the  direction  of  reducing  the 
number  of  typical  B.  coli.  This  constitutes  an  intrinsic 
difference  between  the  bacterial  flora  of  the  raw  and  filtered 
waters,  and  as  the  typical  B.  coli  are  considered  specially 
significant  of  undesirable  excremental  pollution,  it  is  a 
difference  which  may  mean  far  more  than  the  actual  figures 
seem  to  indicate.  The  processes  which  affect  this  modifica- 
tion of  the  original  biological  attributes  of  the  raw  waters  may 
operate  far  more  powerfully  in  the  direction  of  eliminating 
the  microbes  of  epidemic  disease  "  {Fifth  Annual  Report,  p.  12). 
It  should  also  be  noted  that  Houston  uses  glucose,  lactose, 
and  saccharose  gelatin  media.  His  statement  on  this  point  is 
as  follows  :  "  Particular  attention  must  again  be  directed  to 
the  value  of  these  solid  gelatin  sugar  media.  Times  without 
number  a  microbe  which  failed  to  show  any  visible  develop- 
ment of  gas  in  liquid  sugar  media,  has  been  found  to  give 
abundant  gas  when  grown  in  solid  gelatin  media  containing 
the  same  sugars." — First  Report  on  Research  Work,  p.  7). 

These  media  are  described  on  page  49  of  the  Report  on  the 
Metropolitan  Water  Supply  for  January,  1907.  They  have 
the  following  composition  : — 

Lemco  . .  . .  . .  1  per  cent. 

Peptone       . .  . .  . .  . .     2  ,, 

Gelatin         . .  . .  . .  . .     7*5 

KOH  . .         1  c.c.  of  a  5  per  cent  solution. 

Add  1  per  cent  of  glucose,  lactose,  or  saccharose.  The 
glucose  tubes  are  not  tinted,  the  lactose  tubes  are  tinted  with 
litmus,  and  the  saccharose  tubes  with  neutral-red. 

After  inoculation,  such  tubes  are  placed  for  exactly  three 
hours  in  the  blood-heat  incubator.  This  melts  the  gelatin, 
allows  of  some  multiplication  of  the  organisms  to  take  place, 
and  helps  to  effect  their  distribution  throughout  the  medium. 
The  tubes  are  then  placed  in  the  ice  chest  for  half  an  hour  to 
allow  the  gelatin  to  set,  and  thereafter  incubated  for  twenty- 
four  hours  at  20°-22°  C.  Abundant  gas  formation  (in  a  posi- 
tive result)  is  visible  long  before  the  twenty-four  hours  are 
completed. 

In  such  a  medium  liquefaction  of  gelatin  can  also  be  noted. 

IV.  Streptococci. — To  10  c.c.  of  MacConkey's  glucose 
broth,  quadruple  strength,  add  50  c.c.  of  sample  water,  and 


SPECIAL    EXAMINATIONS  359 

incubate  at  37 °  C.  for  twenty-four  hours.  Examine  a 
hanging  drop,  and  if  cocci  are  seen,  make  a  subculture  by 
smearing  a  platinum  loopful  of  culture  over  a  plate  of 
Drigalski  and  Conradi's  medium  (nutrose,  -lactose,  -agar). 
Incubate  at  370  C.  for  twenty-four  hours,  and  then  sub- 
culture all  the  minute  colonies  into  tubes  of  Houston's 
Lemco  medium  (see  p.  222),  containing  0-5  per  cent  of 
lactose,  mannite,  raffinose,  saccharose,  and  salicin  respect- 
ively, and  also  into  milk.  Incubate  for  two  days  at  370  C, 
and  observe  results. 

From  various  researches,  extending  over  ten  years,  into 
the  characteristics  of  faecal  streptococci  (see  Fifth  Research 
Report,  and  other  references  there,  p.  13),  Houston  has 
found  that  of  100  "  sewage  works  "  streptococci — 

All  produced  acid  in  lactose  and  raffinose  media. 

All  clotted  milk. 

None  reduced  nitrates  to  nitrites. 

All  but  three  produced  acid  in  salicin  medium. 

Forty-nine  produced  acid  in  saccharose  medium. 

Only  four  produced  acid  in  mannite  medium. 

These  reactions  differ  from  those  given  by  Andrewes  and 
Horder  for  streptococcus  faecalis  (see  p.  224). 

In  1909,  out  of  156  samples  of  raw  river  water  (Thames, 
Lee,  and  New  River,  52  from  each),  28  were  found  to  contain 
streptococci  in  1  c.c. ;  1908  subcultures  were  made,  and  from 
these  71  streptococci  were  isolated,  plus  3  from  o*i  c.c.  of 
water,  making  71  -f-  30,  or  101  in  all,  or  say  1 11,  so  as  to  over- 
state any  error.  That  is,  in  lactose-positive  streptococci 
were  found  in  156  c.c.  of  raw  river  water,  which  amount 
would  yield  of  all  bacteria  on  gelatin  plate  over  two  million 
colonies  ;  on  agar  plate  about  sixty  thousand  ;  and  on  bile- 
salt  agar  about  six  and  a  half  thousand.  The  ratio,  therefore, 
of  streptococci  to  total  bacteria  (growing  on  gelatin  plate)  in 
the  raw  river  water  works  out  at  one  to  twenty  thousand. 
The  type  of  streptococcus  usually  met  with  in  these  156 
samples,  Houston  describes  as  the  la-mi-ra-sac-sal  variety 
(i.e.  acid  in  lactose,  clot  in  milk,  acid  in  raffinose,  saccharose, 
and  salicin  media).  As  noted  above,  of  the  faecal  streptococci 
(100)  examined  by  him,  97  clotted  milk,  and  produced  acid  in 
lactose,  raffinose,  and  salicin;  and  of  these  97,  48  produced 
acid  and  49  no  appreciable  change  in  a  saccharose  medium. 

In  other  researches  he  found  that  in  human  faeces  of  the 


360         PUBLIC    HEALTH    BACTERIOLOGY 

streptococci  isolated,  15  per  cent  were  of  the  lamirasacsal 
variety,  whereas  in  cow-dung  58  per  cent  of  those  isolated 
were  of  this  variety. 

Houston  gives  thus  his  chief  reasons  for  considering  the 
streptococcus  test  of  value  in  the  examination  of  water 
supplies  : — 

1.  That  streptococci  are  superabundant  in  human 
faeces. 

2.  That  faecal  streptococci  are  absent  or  non-discover- 
able in  a  relatively  large  volume  of  pure  water. 

3.  That  faecal  streptococci  do  not  multiply  in  pure  water. 

4.  That  some  faecal  streptococci  are  of  feeble  vitality, 
and  that  the  presence  of  such  in  a  water,  if  they  could  be 
differentiated  from  their  more  robust  companions,  would 
seem  to  indicate  pollution  of  recent  and  therefore  of  a 
specially  dangerous  nature.  Dr.  Houston  concludes  that 
human  faeces  usually  contain  a  multitude  of  streptococci, 
100,000  per  gramme  being  an  underestimate  of  the  average 
number. 

V.  Bac.  Enteritidis  Sporogenes. — Inoculate  10  c.c.  of 
sample  water  into  10  c.c.  "  whole  "  sterile  milk.  Heat  to 
8o°  to  850  C.  for  ten  minutes.  Cool  in  running  water,  and 
incubate  anaerobicallyin  aBuchner's  tube  at  37°C.  for  forty- 
eight  hours.  Examine  for  coagulation.  The  curd  is  almost 
completely  separated  from  the  whey,  and  is  torn  by  gas 
formation,  part  gathering  with  the  cream  at  the  top.  The 
whey  is  only  slightly  turbid,  and  contains  numerous  bacilli. 
The  growth  has  the  odour  of  butyric  acid,  and  the  B. 
enteritidis  sporogenes  is  distinguished  from  the  B.  butyricus 
of  Botkin  only  by  its  pathogenicity  when  injected  into  a 
guinea-pig,  which  dies  twenty-four  hours  after,  with  green 
discoloration  and  oedema  at  the  seat  of  entry,  and  there 
may  be  gas  formation,  gangrene,  and  a  disagreeable  odour. 

VI.  Isolation  of  B.  Typhosus. — This  is  a  very  difficult 
procedure  for  various  reasons.  The  most  important  are  : 
The  small  numbers  of  B.  typhosus  usually  present ;  their 
rapid  disappearance  from  sewage-polluted  water,  so  that 
they  are  often  absent  when  fresh  cases  arising  from  a 
polluted  water  occur — that  is,  they  die  out  in  about  the 
same  time  as  the  incubation  period  of  the  disease  ;  and  the 


SPECIAL    EXAMINATIONS  361 

fact  that  the  organism  is  easily  outgrown  by  the  other 
excremental  bacteria,  and  so  is  crowded  out  in  most  media. 
When,  to  help  matters,  some  agent,  inhibitory  to  the 
others,  is  added  to  the  medium,  the  typhoid  organism  is 
likewise  affected,  though  to  a  lesser  degree. 

From  all  these  causes  attempts  at  isolation  of  the  B. 
typhosus  from  water  supplies  usually  fail. 

The  first  step  is  to  concentrate  the  water.  This  can  be 
done  by  : — 

(a.)  Centrifugalization  of  large  quantities,  and 
plating  the  sediment. 

(b.)  Precipitation  by  entanglement  in  a  chemical 
precipitate,  such  as  weak  soda  solution  and  ferrous 
sulphate  added  to  the  water,  or  weak  alum  and 
lime-water  solutions.  Centrifuge,  dissolve  precipi- 
tate in  neutral  potassium  tartrate,  and  plate  on 
solid  media. 

(c.)  Filtration  through  unglazed  porcelain  (Pas- 
teur-Chamberland  bougie) .  Wash  bougie  by  brush- 
ing with  10  c.c.  of  sterile  water,  and  use  product  to 
smear  plates.  The  candle  should  filter  from  without 
inwards.  Bacteria  are  apt  to  be  lost  in  the  pores  of 
the  filter.  Some  filter  from  within  outwards,  add 
broth,  and  thus  get  a  primary  culture. 

(d.)  Evaporation   of   the   bulk   of   the  water   at 

blood-heat  under  reduced  atmospheric  pressure. 

The  enrichment  method  of   Hoffman  and  Ficker  is  to 

add  nutrose,  caffein,  and  crystal  violet  to  the  sample,  to 

make  it  a  suitable  growing  medium  for  B.  typhosus.     For 

exact  proportions  see  page  239. 

The  first  method — that  is,  centrifugalization — is  mostly 
favoured. 

One  hundred  c.c.  of  the  sample  are  centrifuged.  The 
sediment  is  plated  direct  on  to  rebipelagar,  and  incubated 
at  37°  C.  for  twenty-four  hours.  All  the  colourless  colonies 
are  subcultured  on  to  agar  slopes,  and  incubated,  at  370  C. 
for  twenty-four  hours.  Thereafter  subcultures  are  made 
into  gelatin  stab,  litmus  milk,  peptone  water,  and  glucose, 
lactose,  saccharose,  dulcite,  mannite,  adonite,  and  inulin 
media.  A  motile  organism,  giving  negative  results  to  all 
these  reactions  except  litmus  milk,  in  which  it  gives  acid 


362 


PUBLIC    HEALTH    BACTERIOLOGY 


Summary  of  some  Results  of  Water  Examination, 

Artesian  wells  and  springs,  4  to  100  bacteria  per  c.c. 
Ordinary  wells,  100-2000         ,,  ,, 

Rain  and  snow,  vary  greatly,  from  4  upwards. 
River  waters,  vary  greatly  (see  table  on  page  351). 


Examination  of  Various  Waters  (J.  Hume  Patterson). 


Germs  per  cc. 

Kind  of  Water 

B.  C01.1 

Gelatin  at  180  C. 

Agar  at  370  C. 

3rd  Day 

2nd  Day 

Absent  in 

Upland           /  Raw 

440 

422 

50  c.c. 

surface             ( Filtered 

22 

•                2 

IOO  C.C. 

waters             j  Raw 

(Filtered 

980 

15 

I  c.c. 

320 

5 

50  c.c. 

Raw 

51 

12 

— 

Raw 

592 

34 

— 

Waters            ( Raw 
stored             \  Filtered 

402 

6 

I  c.c. 

I02 

4 

IOO  c.c. 

in  reservoirs       ( Raw 

4°5 

16 

1  c.c. 

|  Filtered 

76 

4 

10  c.c. 

Tap  Water  from 

18 

0 

IOO  c.c. 

Burghs 

58 

14 

IOO  c.c. 

(different  sources) 

03 

28 

50  c.c. 

72 

0 

10  c.c. 

132 

94 

I  c.c. 

160 

113 

50  c.c. 

246 

52 

50  c.c. 

610 

40 

I  c.c. 

656 

63 

t  C.c. 

"  Burn  "  water  near  sew- 

Present in 

age  outfall — 

Above  outfall 

22,300 

400 

I   C.C. 

150  yards  below 

16,800 

1,800 

OOI  c.c. 

600        ,,         ,, 

19,600 

1,400 

OI     C.C. 

Tributary  burn 

1,000 

40 

IOO  C.C. 

Sewage    filter    effluent 

entering  burn 

377,000 

64,000 

OOI  C.C. 

SPECIAL    EXAMINATIONS  363 

and  then  permanent  alkalinity,  and  glucose  and  mannite, 
in  both  of  which  it  produces  acid  only,  may  be  considered 
to  be  Bacillus  typhosus.  It  should  then  be  tried  for 
agglutination  in  high  dilutions  of  anti- typhoid  serum. 
Pfeiffer's  reaction  may  also  be  tried  with  a  known  immune 
serum.     (See  p.  191.) 

VII.  Isolation  of  Spirillum  Cholerae.  —  This  has 
already  been  described  on  p.  324. 

Summary  of  Some  Results  :  see  preceding  page. 

BACTERIOLOGICAL     EXAMINATION    OF    AIR. 

Various  methods  are  employed  : — 

1.  Exposure  of  Plates  for  a  Definite  Time. — Incubate  ; 
enumerate  ;    identify. 

2.  Hesse's  Method. — A  glass  cylinder,  18"  X  2",  is  closed 
at  one  end  by  an  indiarubber  cover,  tightly  adjusted. 
The  other  end  is  stopped  with  a  close-fitting  rubber  cork, 
through  which  passes  a  glass  tube,  attached  by  rubber 
tubing  to  a  litre  flask  filled  with  water,  and  which,  in  its 
turn,  is  similarly  attached  to  another  litre  flask,  empty  and 
having  its  outlet  tube  clamped.  Along  the  bottom  of  the 
cylinder,  50  c.c.  of  nutrient  gelatin  are  spread,  or  may  be 
rolled  all  over  the  inside  of  tube.  The  whole  is  rested  on  a 
tripod  stand.  Sterilize  in  the  usual  way  and  keep  to  see  if 
sterile.  To  test  air :  pierce  a  small  hole  in  rubber  cover, 
and  open  pinchcock  on  empty  litre  flask,  which  place  lower 
than  other.  Water  begins  to  run  from  the  upper  flask  to 
the  lower,  and  aspirates  air  through  the  pierced  hole  over 
gelatin.  By  reversing  the  flasks,  another  litre  of  air  can 
be  aspirated,  and  so  on.  Finally,  detach  the  tubing  from 
cylinder,  cover  the  ends  with  sterile  wool,  and  incubate  for 
twenty-four  hours  onwards. 

This  method  has  been  largely  superseded  by  simpler  ones. 
All  the  microbes  may  not  be  caught  on  the  gelatin  surface. 

3.  Frankland's  Method. — A  tube,  5"  X  J",  containing 
two  plugs  of  glass  wool.  Aspirate  a  known  quantity  of 
air,  remove  the  wool,  and  add  to  nutrient  gelatin,  and  plate. 
The  glass  wool  mixes  with  the  gelatin. 

4.  Petri's  Method. — Like  Frankland's,  only  using  sand 
instead  of  glass  wool.  There  is  apt  to  be  some  difficulty 
in  distinguishing  between  colonies  and  grains  of  sand. 


364 


PUBLIC    HEALTH    BACTERIOLOGY 


5.  Sedgwick  and  Tucker  s  Method.— &  specially  shaped 
tube  is  used,  in  which  are  placed  cotton  plugs  (3),  and  cane 
sugar  is  packed  into  a  narrow  part  of  the  tube.  One  plug 
is  removed,  and  air  aspirated  through  the  sugar  (the  whole 
apparatus  having  been  previously  sterilized  at  1200  C). 
The  plug  is  then  replaced,  the  sugar  is  shaken  down  into 
a  wider  part  of  the  tube,  and  liquid  gelatin  is  poured  in. 
The  sugar  melts  into  the  gelatin,  and  a  roll  culture  is 
made  before  the  latter  solidifies.  Incubate  at  220  C.  and 
count  colonies  daily. 

Organisms  found. — Yeasts,  Spores  of  moulds,  Strepto- 
coccus brevis,  B.  coli,  B.  mycoides,  B.  enteritidis  sporogenes, 
Streptococcus  epidermidis,  Streptococcus  salivarius. 

The  last  two  serve  to  indicate  pollution  by  particles  of 
skin  and  saliva.  The  preceding  ones  indicate  pollution 
by  dirt  from  the  street,  brought  in  on  the  boots,  if  the 
air  tested  is  that  of  a  room  ;   or  it  may  be  blown  in. 

The  effect  of  stirring  dust  is  to  increase  the  ratio  of 
bacteria  to  moulds,  whereas  in  still  air,  bacteria  settle 
down  much  more  rapidly  than  moulds.  The  purer  the 
air,  the  more  nearly  do  the  numbers  of  the  bacteria  and 
of  the  moulds  approximate.  This  is  well  shown  in  the 
figures  found  by  the  examination  of  1  cubic  metre  of  air 
(1000  litres  or  220  gallons),  taken  in  Paris  and  the  suburbs, 
in  the  various  seasons. 


- 

Montsouris. 

Centre  of  Paris. 

Moulds. 

Bacteria. 

Moulds. 

Bacteria. 

Winter 

Spring         

Summer 
Autumn 

145 

195 
245 
230 

170 
295 
345 
195 

1345 

2275 
2500 

2185 

4305 

8080 

9843 
5665 

Particulate  pollution  of  the  air — by  material  from  the 
upper  respiratory  passages  from  the  skin  and  from  the 
street — may  all  be  detected  by  exposing  plates  filled  with 
broth  to  the  air  for  a  definite  time  ;  incubating  anaerobi- 
cally  for  forty-eight  hours  at  370  C,  and  examining  for 
certain  test  organisms.     (Gordon.) 


SPECIAL    EXAMINATIONS  365 

BACTERIOLOGICAL    EXAMINATION     OF     SOIL. 

A  quantity  of  soil  may  be  collected  in  a  platinum  or 
metal  spoon,  or  in  a  sterile  tin  trough,  or  by  a  Fraenkel's 
borer.  A  definite  quantity  is  ground  up  in  a  sterile  mortar 
and  then  mixed  with  known  quantities  of  sterile  water. 
Cultures  are  then  made  from  definite  quantities  of  these 
dilutions,  and  the  number  of  B.  coli,  of  streptococci,  and 
of  spores  of  B.  enteritidis  sporogenes,  are  determined  and 
returned  as  so  many  per  gramme  of  the  original  soil. 
Houston  found  100,000  (of  all  bacteria)  per  grm.  in  sandy 
uncultivated  soil,  and  1,500,000  per  grm.  in  garden  soil. 
Spores  are  determined  by  adding  1  c.c.  of  a  dilution  to- 
10  c.c.  of  gelatin  ;  heat  to  8o°  C.  for  ten  minutes  to  destroy 
non-sporing  bacteria,  plate,  incubate,  and  count  as  late  as 
liquefaction  (if  present)  will  allow.  The  examination 
for  specific  pathogenic  bacteria  is  carried  out  by  injecting 
animals  subcutaneously,  with  some  of  the  material  or  a 
dilution  of  it  in  normal  saline  (o-8  per  cent). 

Organisms  Found. — The  bacteria  found  in  soil  are  : — 
(a.)  Putrefactive  organisms — 

B.  mycoides  (earth  bacillus). 
B.  subtilis  (hay  bacillus). 
B.  megatherium. 
(b.)  Nitrifying  organisms  or  nodule  bacteria — 
Nitroso-bacteria,  or  nitrite  formers. 
Nitro-bacteria  or  nitrate  formers. 
(c.)  Pathogenic  organisms — 
Bacillus  of  tetanus. 

,,       of  malignant  oedema. 
„       of  quarter  evil. 

enteritidis  sporogenes. 


DUST,     SEWAGE     AND     SEWAGE     EFFLUENTS, 
AND     EXCREMENTAL    MATTERS. 

These  are  examined  on  similar  principles,  except  that  a 
dry  solid,  like  dust,  may  be  weighed  out,  crushed  in  a  sterile 
mortar,  and  made  into  a  primary  dilution  with  distilled 
water.  Gordon  found  that  in  the  dust  collected  at  air  inlet 
at  House  of  Commons  there  were  under  10  bacteria  per 


366         PUBLIC    HEALTH     BACTERIOLOGY 

grm.  ;  in  debating  chamber  100,000  per  grm.  (B.  coli 
1000,  streptococci  10,  B.  enteritidis  sporogenes  1000)  ;  in 
the  division  lobby  1,000,000  per  grm.  (containing  1000  of 
each  of  B.  coli,  streptococci,  and  B.  enteritidis  sporogenes)  ; 
and  in  New  Palace  Yard,  100,000  per  grm.,  of  which  10,000 
Were  B.  coli,  under  1000  streptococci,  and  100  B.  enteritidis 
sporogenes). 

MILK. 

In  a  healthy  cow  the  milk  within  the  udder  is  sterile. 
In  the  milk  ducts  and  teats  a  certain  number  of  bacteria 
may  be  found,  even  in  healthy  animals.  From  the  position 
of  the  udder  and  the  mode  of  milking,  it  is  not  possible 
to  collect  milk  under  aseptic  precautions,  and  in  the 
most  favourable  circumstances  freshly  taken  milk  in  the 
pail  will  yield  100  to  500  bacteria  per  c.c.  Under  ordinary 
conditions  the  yield  is  much  greater,  varying  from  2000 
to  6000  per  c.c  where  less  care  is  used,  and  with  careless 
manipulation  (the  usual  method),  30,000  to  100,000  per  c.c. 
With  such  a  bacterial  content  from  the  beginning,  it  is  not 
surprising  that  at  summer  temperatures  the  count  in 
twenty-four  hours  should  be  enormous,  reaching  into 
millions  and  even  hundreds  of  millions  per  c.c.  The 
species  found  include  almost  all  known  varieties. 
Swithinbank  and  Newman  describe  120  "  milk  bacteria," 
apart  from  organisms  of  water,  soil,  etc.  The  varieties 
found  are  there  largely  because  of  their  presence  in  the 
environment,  or  their  power  to  outgrow  other  species  under 
the  cultural  conditions.  Pathogenic  bacteria  are  found  in 
milk  either  (1)  derived  from  the  cow,  e.g.,  tubercle 
bacillus,  actinomyces,  anthrax  bacilli,  streptococci,  foot  and 
mouth  disease  germ  ;  or  (2)  from  the  introduction  into  it 
of  infectious  material  of  human  origin,  e.g.,  typhoid,  diph- 
theria, germ  of  scarlatina,  cholera ;  or  (3)  from  the  air,  when 
kept  in  unsuitable  conditions.  Under  (2),  the  milking  of 
cows,  or  the  handling  of  milk  by  persons  suffering  from 
disease  or  by  acute  or  chronic  carriers  of  disease  germs, 
the  use  of  infected  water  to  rinse  cans,  and  the  pollution  by 
flies,  are  the  main  acting  causes.  The  milk  of  infected 
goats  contains  the  germ  of  Malta  fever.  Besides  these, 
milk  contains  many  faecal  organisms  derived  from  the 


SPECIAL    EXAMINATIONS  367 

soiled  and  uncleansed  skin  near  the  udder,  and  from  the 
dust  of  the  byre. 

Suggested    Bacteriological     Standard    (Newman). — 
(a.)  Acidity  of  ioo  c.c.  +  2  ex.  phth.  (o-i  per  cent)  to 
be  not  more  than  25  c.c.  N/10  alkali. 
(b.)  No  excess  of  blood  or  pus  cells. 
(c.) I  No    B.   coli,    B.    enteritidis    sporogenes,     nor    B. 
enteritidis  (Gaertner)  in  1  c.c. 
(d.)  The  milk  to  be  non- virulent. 

The  New  York  Milk  Commission  specify  that  certified 
milk  shall  contain  not  more  than  30,000  bacteria  per  c.c, 
and  in  Philadelphia,  the  Commission  standard  is  not  more 
than  10,000  per  c.c,  and  little  difficulty  has  been  experienced 
in  conforming  to  this  test.  Boston  has  fixed  a  limit  for 
ordinary  milk  of  500,000  per  c.c,  Milwaukee  of  250,000, 
and  Rochester,  N.Y.,  of  100,000  per  c.c.  The  average 
content  in  this  country  is  400,000  per  c.c,  as  sold. 

Microscopic  Examination. — Shows  round  oil  globules 
and  a  little  epithelium.  Abnormal  constituents  are : 
much  epithelium,  pus  cells,  conglomerate  masses  and 
casts  of  lacteal  tubes,  and  colostrum. 

Quantitative  Examination. — Decimal  mode  of  dilution. 
Add  9  c.c  of  water  to  each  of  a  number  of  test  tubes,  plug, 
and  sterilize. 

Add  1  c.c.  of  milk  sample  to  No.  1  tube  : 

dilution  =  1-10,  or  1  c.c  =  o-i  c.c  milk. 
Add  1  c.c.  of  No.  1  tube  to  No.  2  tube  : 

dilution  =  1-100,  or  1  c.c.  —  o-oi  c.c  milk. 
Add  1  c.c.  of  No.  2  tube  to  No.  3  tube  : 

dilution  =  1-1000,  or  1  c.c.  =  o-ooi  c.c  milk. 
Add  1  c.c.  of  No.  3  tube  to  No.  4  tube : 

dilution  =  1-10,000,  or  1  c.c.  —  o-oooi  c.c  milk. 

And  so  on  for  any  number  of  dilutions,  mixing  well  each 
time,  with  sterile  pipette. 

Plate  1  c.c  of  the  various  dilutions  on  gelatin  and 
agar,  and  count  colonies  in  twenty-four  to  forty-eight 
hours.  (Begin  plating  with  weakest  dilutions,  if  the 
same  pipette  is  to  be  used  throughout.) 


368         PUBLIC     HEALTH     BACTERIOLOGY 

Qualitative   Examination. — 

i.  For  Faecal  Organisms. 

Inoculate  tubes  of  MacConkey's  bile-salt  glucose  peptone 
(10  c.c.)  with  i  c.c.  from  each  dilution,  beginning  with  the 
weakest  (i  cc=o-oooooi  c.c.  milk,  or  one-millionth),  and 
continuing  in  reverse  order.  Also  one  tube  with  i  c.c.  of 
undiluted  milk,  and  one  tube  of  double  strength  broth 
(MacConkey's)  with  10  c.c.  of  undiluted  milk.  Incubate 
for  two  days  at  370  C.  All  the  cultures  showing  acid  and 
gas  are  subcultured  as  detailed  under  Water  (page  353). 

2.  For  B.  Enteritidis  Sporogenes. 

Inoculate  tubes  containing  15  c.c.  sterile  whole  milk,  with 
o-ooi  c.c,  o-oi  c.c,  o-i  c.c,  1  c.c,  10  c.c  and  a  tube  con- 
taining 30  c.c.  with  100  c.c  Heat  to  &a°  C.  for  10  minutes 
for  the  smaller  amounts,  and  20  minutes  for  the  larger. 
Incubate  anaerobically  at  37°  C.  for  2  days,  and  observe 
for  the  "  enteritidis  change  or  reaction  "  (page  319). 

3.  For  Tubercle  Bacilli. 

Centrifugalize  and  take  sediment,  make  a  film,  fix, 
clear  with  ether  and  alcohol,  and  stain  for  acid-fast 
bacilli.  Failure  to  find  them  is  inconclusive  ;  on  the 
other  hand,  all  acid-fast  forms  found  are  not  tubercle 
bacilli.  In  case  of  failure,  or  of  success  to  prove  nature, 
use  the  animal  experiment.  Centrifuge  250  c.c.  of  milk, 
add  sterile  water  to  sediment,  and  inject  into  a1  guinea-pig 
intraperitoneally.  The  animal  is  killed  in  three  weeks  (if 
it  has  not  died  before  that  time  from  septicaemia  or  severe 
infection),  and  the  carcase  sought  for  typical  lesions. 
Among  acid-fast  organisms,  the  forms  found  in  hay,  butter, 
etc.,  are  slightly  pathogenic,  but  are  easily  distinguished  by 
their  rapid  growth  on  cultivation.  An  attempt  at  cultiva- 
tion from  the  lesions  in  the  guinea-pig  should  therefore  be 
made. 

The  following  excerpt  from  Dr.  J.  Hume  Patterson's 
Report  for  1910,  to  the  County  M.O.H.,  Lanarkshire, 
details  some  experiments  on  the  distribution  of  the  tubercle 
bacilli  in  the  "  fore,"  "  mid,"  and  "  strippings  "  milk  : — 


SPECIAL    EXAMINATIONS  369 

Tubercular  Disease  of  the  Udder  of  Cows. — Milk. — During 
the  year  (1910)  227  samples  of  milk  from  225  cows  with 
suspected  disease  of  the  udder  were  examined,  53  of  which 
gave  positive  results,  giving  an  average  of  23-5  per  cent. 
From  the  beginning  of  February,  special  attention  has  been 
given  to  the  examination  of  smear  preparations,  in  order  that 
an  immediate  report  might  be  given,  instead  of  waiting  three 
weeks  for  the  animal  inoculation  test.  Out  of  44  specimens 
so  examined,  31  were  found  positive.  The  milk  in  each  case 
was  stopped  within  two  days  of  the  taking  of  sample. 

It  might  here  be  mentioned  in  connection  with  the  examina- 
tion of  smear  preparations,  that  an  elaborate  and  complicated 
process  of  staining  is  not  necessary,  as  a  good  result  does  not 
depend  so  much  on  the  process  of  staining,  as  the  time  spent 
on  the  examination  of  the  film.  The  method  now  adopted 
is  the  ordinary  Ziehl-Neelson,  the  film,  after  discoloration, 
being  well  washed  in  absolute  alcohol  to  clear  it  up. 

All  the  samples  giving  negative  smears  were  subjected  to 
the  animal  inoculation  test. 

Twenty-seven  samples  of  milk  were  obtained  from  four  of 
the  positive  cows,  for  the  purpose  of  ascertaining  whether 
the  "  fore,"  "  mid,"  or  "  strippings  "  milk  contained  the 
most  bacilli,  also  whether  the  bacilli  were  more  numerous  in 
the  cream  than  in  the  deposit.  The  samples  were  taken  by 
the  County  Veterinary  Surgeon. 

In  each  case  the  whole  of  the  milk  from  the  affected  quarter 
was  drawn  off  in  separate  consecutive  samples,  the  last  one 
always  containing  the  last  drop  of  milk  procurable. 

Each  specimen  when  received  was  centrifugalized  for  15 
minutes,  and  smear  preparations  made  with  one  platinum 
loopful  of  the  cream  and  of  the  deposit.  The  loopful  was 
then  thoroughly  mixed  on  the  slide  and  spread  over  an  area 
the  breadth  of  the  slide  and  one  inch  in  length.  The  films 
were  stained  by  Ziehl-Neelson's  method.  The  count,  which 
of  course  was  an  approximate  one,  was  carried  out  by  running 
the  tl  oil  immersion  lens  straight  across  the  centre  of  the 
film,  counting  the  bacilli  in  each  field  and  multiplying  the 
total  by  twelve.  (The  film  being  an  inch  in  length  and  the 
lens  used  ^).  Where  the  bacilli  were  few  in  number,  the  whole 
film  was  examined  and  counted.  The  results  obtained  were 
as  shown  in  the  table  on  following  page. 

It  will  be  seen  from  the  results  obtained  from  Cows  I.,  II., 
and  IV.,  that  the  "  mid  "  milk  was  richest  in  bacilli,  and 
would  evidently  be  the  proper  part  to  take  when  such  a  milk 
was  to  be  examined,  more  especially  by  smear  preparation. 
No  reliance  could  be  placed  on  the  samples  taken  from  Cow 
No.  III.,  as  the  milk  was  too  small  in  amount. 

24 


370 


PUBLIC    HEALTH    BACTERIOLOGY 


Cow  No.    I. —                          Specimen 

Cream 

Deposit 

1  •  • 

2  .  . 

not  done 

negative 

3 

'8  bac. 

4 

14       M 

5 

negative 

6 

6  bac. 

7 

2      ,, 

8 

8      „ 

Cow  No.  II. — 

i     Slight  cream,  J"  deposit    . . 

7  bac. 

660  bac. 

2            »                  i* 

8      „ 

912      „ 

3            »                  i"        „         •• 

3              „ 

1,380      „ 

4          m               £" 

5      >, 

3,8i6      „ 

5     Very  slight  cream,  £"  deposit 

5      » 

2,196      „ 

6          „               „            |*       „  .  . 

4          M 

636      „ 

7          »               „            i"       „  •  ■ 

2        „ 

1,104      „ 

8     Very  watery.     Thin  layer  of 

cream,  i"  pus-like  deposit 

0        ,, 

396      „ 

Cow  No.  III. — 

i     Watery,  slight  cream,  \"  pus- 

like deposit 

o 

168  bac. 

3     Ditto         ditto         \"  ditto 

o 

192 

4     Ditto         ditto         ¥  ditto 

o 

252      „ 

(Milk  from  quarter  only  amounted  to  about 

2  ozs.) 

Cow  No.  IV. — 

i     Watery,  slight  cream,  £"  deposit 

120  bac. 

5,172  bac. 

r    ,. 
r    ., 

very    slight    cream,    |" 
deposit 

heavy  cream,  -^"  deposit 
¥     blood- 
stained deposit 


1,020 
1,212 

1,584 

364 
2,532 

1,824 


6,804 

5,9io 

14,460 

10,908 
5.316 

4.824 


From  the  samples  taken  from  Cows  II.,  III.,  and  IV.,  it 
was  clearly  shown  that  the  deposit  is  the  proper  part  of  the 
specimen  to  take  for  the  examination,  it  containing  by  far 
the  greater  number  of  tubercle  bacilli. 

4.  For    Actinomyces. 

Make  a  film  from  sediment,  and  stain  by  acid-fast 
method.  Actinomycosis  in  the  udder  of  the  cow  is 
usually  alleged  to  be  rare,  but  is  stated  to  be  more 
common  in  the  sow.  In  an  article  on  "  The  Occurrence 
of  Actinomycosis  in  Cows'  Udders,"  by  Dr.  J.  Hume 
Patterson,  in  the  Journal  of  Meat  and  Milk  Hygiene  (vol.  i, 


SPECIAL    EXAMINATIONS  371 

No.  i,  Jan.  191 1),  the  writer  cites  evidence  which  goes  to 
show  that  it  may  not  be  so  uncommon.  Out  of  fifty 
specimens  from  different  udders  submitted  to  him  for 
suspected  tubercle,  in  five  cases  the  lesions  proved  to  be 
actinomycotic.  The  lesions  in  each  case  were  indistinguish- 
able by  the  naked  eye  from  those  of  tuberculosis.  On 
cutting  into  the  substance  of  the  udder  numerous  cream- 
coloured  foci,  similar  to  tubercles,  were  seen,  ranging  from 
the  size  of  a  pin-head  to  that  of  a  pea.  The  part  affected 
was  also  of  a  brownish  tint,  as  is  so  often  seen  in  tubercu- 
losis of  this  tissue.  Smear  preparations  showed  elements 
of  actinomyces  in  four  of  the  cases  ;  in  one,  no  elements 
were  found,  but  on  making  sections  typical  actinomyces 
were  found.  All  the  others  were  confirmed  by  making 
paraffin  sections.  In  one  of  the  smears  both  tubercle 
bacilli  and  actinomyces  were  found  ;  and  if  not  on  one's 
guard,  such  a  case  could  be  readily  dismissed  as  tuber- 
culosis without  looking  for  actinomycosis.  Dr.  Patterson 
says  :  "I  am  confident  from  my  experience  in  these  cases 
that  if  each  suspected  tubercular  udder  were  subjected  to 
a  microscopical  examination,  the  percentage  of  actinomy- 
cotic udders  would  be  greater  than  is  generally  supposed. 

In  regard  to  milk,  many  of  the  samples,  taken  by  veteri- 
nary surgeons  inspecting  dairy  herds  from  cows  having 
what  appeared  to  be  marked  tubercular  lesion  of  the  udder, 
have  proved  negative  even  on  animal  inoculation.  Dr. 
Patterson  asks  the  question  :  "  Might  these  not  be  cases 
of  actinomycosis  ?  "  and  cites  the  following  case  : — 

"  During  last  winter's  inspection  (1909-10)  a  case  occurred 
where  the  lesion  of  the  udder  was  markedly  nodular  and 
similar  to  tubercle.  A  sample  of  the  milk  was  taken,  and  a 
guinea-pig  inoculated  with  negative  result.  Not  satisfied 
with  this  result,  samples  were  again  taken  from  all  four 
quarters  of  the  same  cow.  These  samples  again  proved 
negative  on  animal  inoculation.  Smear  preparations  from 
these  last  samples,  made  from  the  deposit  of  the  centrifugalized 
milk,  showed  a  few  acid-fast  rod-shaped,  and  a  few  fragments 
of  club-shaped,  elements  suggestive  of  actinomyces,  in  those 
samples  taken  from  both  hind  quarters  and  from  the  left  fore 
quarter.  I  was  unable  to  procure  the  udder  for  further 
examination,  but  am  convinced  that  this  was  a  case  of 
actinomycosis.     In  actinomycosis  of  the  human  subject,  it  is 


372         PUBLIC    HEALTH    BACTERIOLOGY 

yet  doubtful  how  infection  takes  place,  as  the  cereal  theory- 
has  been  partly  exploded,  through  cases  arising  which  had  no 
connection  with  grain;  and  I  think  it  is  just  possible  infection 
may  be  conveyed  by  the  milk  of  such  a  cow  as  I  have  quoted, 
where  the  elements  of  the  disease  were  found  in  the  fluid. 
If  that  be  so,  this  disease,  as  affecting  the  udder  of  the  cow, 
warrants  more  attention  than  is  given  to  it  at  the  present 
moment  in  connection  with  our  milk  supplies."  In  this  regard 
the  present  writer  was  consulted  in  19 10  by  a  young  man  of 
about  twenty  years,  who  had  recently  returned  to  Scotland 
from  Canada.  He  had  been  treated  in  several  hospitals  for 
tuberculosis.  The  history  being  irregular,  his  spit  was  sent  to 
the  City  Bacteriological  Laboratory  (Glasgow)  for  examination 
with  this  note  :  "If  you  do  not  find  tubercle  bacilli,  look  for 
something  else."  Dr.  Sutherland,  who  examined  the  speci- 
men, found  actinomyces,  and  the  diagnosis  was  confirmed 
later  in  the  Glasgow  Royal  Infirmary.  He  died  some  months 
later,  but  a  post-mortem  examination  was  not  obtained.  The 
history  bearing  on  the  point  at  issue  is  this  :  About  two  years 
previously  he  left  his  position  as  an  office  boy  in  Glasgow,  and 
went  to  near  Calcary,  Alberta,  Canada,  where  he  became  a 
farmer's  boy.  One  of  the  cows  he  had  to  milk  had  a  chronic 
sore  on  its  udder,  and  he  was  warned  to  milk  this  cow  gently, 
so  as  not  to  make  the  sore  bleed.  After  about  one  year  he 
took  ill  at  this  farm  with  a  sore  throat,  followed  by  a  swollen 
right  submaxillary  gland.  The  gland  was  incised  and  healed 
well.  Then  another  swelling  appeared,  and  another,  and  so  on. 
This  certainly  looked  like  a  milk  infection,  but  verification 
was  not  possible. 

5.  For  Johne's  Bacilli. 

The  prevalence  of  Johne's  disease  in  cattle  in  Great 
Britain  being  now  well  established,  the  bacilli  may  find 
their  way  into  the  milk  from  the  diarrhceal  stools  in  the 
earlier  stages  of  the  disease.  The  bacilli  are  shorter  than 
the  tubercle  bacilli,  but  are  equally  acid-fast  and  alcohol- 
fast.     Twort  has  cultivated  them  on  egg  media. 

6.  For  Streptococci. 

Houston  advises  the  use  of  the  medium  of  Drigalski  and 
Conradi  (lactose-nutrose-agar)  in  plates.  The  plates  are 
inoculated  by  smearing  over  the  surface  of  them  o-i  c.c.  of 
each  of  the  dilutions  given  above.    Incubate  twenty-four  to 


SPECIAL    EXAMINATIONS  373 

forty-eight  hours  at  37  °  C,  and  subculture  the  minute  colonies 
formed  into  broth,  and  repeat  cultivation  as  to  time  and  tem- 
perature. Make  films  from  the  broth  and  examine  micro- 
scopically, and  if  found  in  pure  culture,  subject  the  organism 
to  the  differential  tests  for  streptococci  and  to  nitrate  broth 
test.  According  to  Houston,  58  per  cent  of  the  Streptococci 
faecalis  of  the  cow  are  of  the  lamirasacsal  variety  ;  that  is, 
clot  milk  and  ferment  lactose,  rafhnose,  saccharose,  and 
salicin.  It  forms  short  chains,  and  is  not  pathogenic  to 
mice.  The  S.  pyogenes  does  not  clot  milk,  ferments 
lactose,  saccharose,  and  sometimes  salicin,  but  does  not 
ferment  rafhnose,  and  is  pathogenic  to  mice,  forms  long 
chains,  and  does  not  reduce  nitrates. 

Leucocyte  Test. — In  testing  for  chronic  mastitis  in  cows, 
Trommsdorff  found  that  the  deposit  of  leucocytes  after 
centrifugalizing  the  milk  in  a  specially-shaped  tube,  was  a 
good  guide  as  to  the  necessity  for  further  investigation. 
In  an  enquiry  on  these  lines,  he  found  20  per  cent  of  chronic 
mastitis  in  cows,  and  it  was  associated  with  the  presence 
in  very  large  numbers  of  capsulated  streptococci.  Such 
cows  give  less  milk  The  milk  must  be  drawn  directly 
from  the  animal  before  one  can  say  that  the  streptococcus 
is  from  the  udder  of  the  cow. 

Centrifugauzation  of  Milk 
Is  used  to  precipitate  the  gross  dirt,  pus  cells  and 
leucocytes,  and  bacteria.  According  to  Scheurlen,  the 
ordinary  milk  bacteria  in  the  proportion  of  75  per  cent 
of  them  go  into  the  cream,  as  do  also  the  organisms  of 
anthrax,  typhoid,  and  cholera.  The  other  25  per  cent  of 
these  remain  in  the  separated  milk.  On  the  other  hand, 
tubercle  bacilli  are  largely  found  in  the  sediment,  a  few 
only  passing  into  the  cream  and  separated  milk. 

Fermentations. 

Lactic  Acid. — The  development  of  acid  and  curd  occurs 
normally  in  milk  on  keeping  It  is  due  to  the  formation 
of  lactic  acid  from  the  milk  sugar  or  lactose,  by  the  action 
of  enzymes  produced  by  microbic  growth.  Many  species 
of  bacteria  are  able  to  produce  the  lactic  fermentation,  but 
in  milk  those  most  commonly  causing  it  are  :    (1)  Bacillus 


374         PUBLIC    HEALTH     BACTERIOLOGY 

lactis  aerogenes,  and  (2)  Streptococcus  lacticus  (Kruse), 
identical  with  the  B .  a  cidi  lactici  (Leichmann) .  Heinemann, 
who  has  investigated  the  subject,  states  that  the  two 
species  are  ordinarily  present  in  naturally  souring  milk, 
the  former  in  abundance  at  the  beginning,  the  latter 
in  the  later  stages  when  the  acidity  has  reached  a  high 
degree.  The  secret  of  the  regularity  of  the  presence  of 
these  two  species  is  their  power  of  withstanding  a  much 
higher  degree  of  acidity  than  the  other  species  present  at 
the  first.  In  changing  to  lactic  acid,  the  lactose  is  believed 
to  be  first  hydrolyzed  into  glucose  and  galactose. 

CifH.iOu  +  H20  =  C6H1206  +  C6H1206  =  4C3H603 

Coagulation  of  casein  follows  on  acidification  of  the 
milk,  the  amount  of  acid  necessary  to  precipitate  the 
casein  averaging  0-45  per  cent ;  the  terminal  amount  may 
reach  0-85  per  cent.  The  casein  precipitated  by  lactic 
acid  formation  is  never  redissolved,  because  the  high 
acidity  inhibits  the  proteolytic  ferments. 

Casein  precipitation,  however,  may  also  be  due  to  a  non- 
acid  coagulation  caused  by  bacterial  ferments.  Casein 
precipitated  in  this  way  may  be  redissolved  by  a  bacterial 
trypsin  or  casease,  produced  by  the  same  or  other  bacteria, 
and  the  milk  hence  may  become  entirely  liquid,  transparent, 
and  of  a  yellowish  colour. 

B.  bulgaricus  in  milk  culture  produces  2-5  per  cent  of 
lactic  acid,  and  0-05  per  cent  of  acetic  and  succinic  acids, 
is  non-pathogenic,  and  exerts  no  putrefactive  action  upon 
proteids.  Metchnikoff  suggested  its  use  in  milk  cultures 
as  a  food  to  inhibit,  by  its  acid  production,  the  growth  in 
the  intestine  of  the  class  of  bacteria  which  break  up 
proteids,  the  bacteria  of  putrefaction.  This  is  Metchnikoff 's 
bacteriotherapy,  which  has  been  extensively  practised. 

B.  bulgaricus  is  a  large,  non-motile,  non-sporing,  Gram- 
positive  bacillus,  with  square  ends  (like  B.  anthracis).  It 
forms  short  and  long  chains.  It  shows  little  or  no  growth 
on  ordinary  media  or  below  370  C.  Optimum  temperature  : 
42 °  C.  Grows  in  dextrose-peptone  broth,  to  which  calcium 
carbonate  has  been  added. 

Butyric  Acid  fermentation  of  milk  occurs  occasionally 
in  milk  from  the  growth  of  anaerobic  bacteria.     It  is  a 


SPECIAL    EXAMINATIONS  375 

much  slower  process  than  the  lactic  one,  and  can  also  be 
produced  by  some  pathogenic  anaerobes,  e.g.,  bacilli 
of  quarter-evil,  malignant  oedema,  B.  Welchii  and  B. 
enteritidis  sporogenes  (Klein). 

Alcoholic  Fermentation  of  milk  occurs  spontaneously 
on  rare  occasions.  The  process  is  due  to  the  natural  in- 
troduction of  yeasts,  and  once  started  can  be  kept  going 
by  infecting  fresh  milk.  Koumiss  is  thus  made  by  the 
Tartars  from  mare's  milk.  Kefir  is  an  effervescent  sour 
milk  made  from  cow's  milk  by  the  addition  of  "  kefir 
grains,"  little  cauliflower-like  excrescences  whose  fermen- 
tative power  is  due  to  Saccharomyces  mycoderma.  Mare's 
milk  is  more  suitable  for  the  preparation,  because  the 
lactic  fermentation  also  accompanies  the  other,  and  the 
duration  of  the  double  fermentation  is  conditioned  by  the 
amount  of  sugar,  which  is  5-5  per  cent  in  mare's  milk  and 
4-8  per  cent  in  cow's.  The  latter  is  richer  in  casein  and  fat, 
and  both  of  these  constitute  disadvantages,  so  that  it  is 
usually  diluted  in  making  koumiss.  In  the  making,  the 
milk  is  stirred  constantly  by  day  but  rested  at  night.  The 
amount  of  alcohol  in  all  these  products — koumiss,  kefir,  and 
cow's  milk  koumiss — is  under  2  per  cent,  and  the  Tartar  is 
capable  of  consuming  three  to  four  gallons  of  such  milk 
on  a  hot  summer's  day  without  becoming  more  than 
hilarious,  and  with  no  digestive  disturbance.  They  have 
been  much  used  as  "  consumption  cures." 

Diseases  of  Milk. 

Unusual  or  abnormal  changes  in  milk  are  sometimes 
referred  to  as  "  diseases."  They  are  produced  by  bacteria 
which  have  got  into  milk  in  various  ways.  Blue,  green, 
and  yellow  milks  are  due  respectively  to  the  bacilli  cyano- 
genes,  erythrogenes,  and  synxanthus  ;  and  red  milk  to 
B.  prodigiosus.  Bitter  milk  is  due  to  a  number  of 
species,  yeasts  and  diplococci  having  been  isolated. 
Slimy  or  ropy  milk  has  been  traced  to  B.  lactis  viscosus, 
said  to  be  a  water  organism.  Slimy  milk  is  produced 
at  Edam  (Holland)  by  the  use  of  a  streptococcus,  for 
the  manufacture  of  Edam  cheese.  Soapy  milk  is  due  to  a 
micrococcus  derived  from  fodder. 


376         PUBLIC    HEALTH    BACTERIOLOGY 

BUTTER. 

Butter  is  made  from  the  cream  of  milk  by  churning  or 
agitation,  whereby  the  globules  of  fat  are  broken,  or 
rather  have  their  casein  envelopes  ruptured,  and  then  the 
fat  globules  adhere.  The  cream  is  first  allowed  to  sour, 
otherwise  the  butter  will  be  flavourless.  The  souring 
is)  brought  about  by  bacteria,  nearly  all  of  which  are  lactic 
acid  formers.  Tuberculosis  is  the  only  infective  disease 
transmitted  by  butter.  Tubercle  bacilli  have  been  found 
alive  and  virulent  in  butter  after  having  been  kept  in  a 
refrigerator  for  five  months.  Rabinowitch's  acid-fast  butter 
bacillus  is  easily  distinguished  culturally  from  the  tubercle 
bacillus.  Foot  and  mouth  disease  has  been  reported  as 
having  been  transmitted  by  butter.  Typhoid  infection 
is  unlikely,  and  has  not  yet  been  definitely  traced. 

CHEESE. 

Cheese  is  the  precipitated  casein  of  milk,  the  casein 
or  curd  being  insoluble.  In  hard  cheeses  the  whey  is 
better  expressed  than  in  soft  cheeses,  and  so  the  sub- 
sequent '  ripening  "  which  is  due  to  bacterial  growth  is 
less  in  the  former  than  in  the  latter.  In  the  ripening  of  the 
curd,  three  groups  of  bacteria  are  engaged,  (i)  acid  pro- 
ducers, like  B.  acidi  lactici,  (2)  casein  digesters,  which  break 
down  the  curd,  and  (3)  gas  producers,  which  honeycomb  it. 
The  actual  organisms  engaged  have  been  determined  in  the 
case  of  particular  cheeses,  and  are  :  a  bacillus  resembling 
the  Bacillus  subtilis,  a  mould  (Oidium  lactis),  a  penicillium 
mould,  and  yeasts.  Tubercle  bacilli  of  the  bovine  and 
human  types  have  been  found  in  cheeses. 

SHELLFISH. 

For  Houston's  methods,  see  Journal  of  Hygiene,  vol.  iv, 
No.  2,  p.  185. 

WATERCRESS  AND  OTHER  VEGETABLES. 

See  Report  to  the  L.C.C.,  by  Houston,  1905. 


SPECIAL    EXAMINATIONS  377 

DISINFECTANTS. 

A  disinfectant  or  germicide  is  a  substance  which  destroys 
the  microbic  causes  of  disease.  The  same  substance 
in  a  weaker  strength  may  act  as  an  antiseptic,  that  is  an 
agent  which  restrains  or  checks  the  growth  of  bacteria 
without  destroying  them.  A  deodorant  is  a  substance 
which  destroys  or  masks  the  offensive  effluvia  or  vapours 
resulting  from  bacterial  growth,  and  may  or  may  not 
have  an  action  on  the  bacteria  themselves.  The  best 
example  of  an  agent  capable  of  all  these  functions  is 
potassium  permanganate,  which  in  strengths  of  5  per  cent 
and  over  is  a  disinfectant,  under  5  per  cent  is  an  anti- 
septic, and  in  all  strengths  is  a  deodorant.  (Solubility, 
1-18  of  water ;  1-3  boiling.)  The  mode  of  action  of 
disinfectants  varies.  Some,  like  the  strong  acids,  char  or 
carbonize  the  bacterial  body  by  oxidizing  the  other 
elements  present.  Others  coagulate  the  bacterial  proto- 
plasm ;  while  some,  diffusing  through  the  cell  wall,  exert 
a  poisonous  action  on  the  protoplasm.  Antiseptics  like 
sugar,  probably  act  by  osmotic  pressure  through  the  cell 
wall,  causing  a  flow  of  water  out  of  the  cell,  thus  drying 
up  the  protoplasm  and  rendering  the  bacterium  inert  for 
the  time  being. 

The  chief  disinfectants  are  the  metallic  salts,  acids 
bases,  the  halogen  elements,  oxidizing  agents,  alcohols 
phenols,  aldehydes,  and  the  essential  oils.  The  salts, 
acids,  and  bases  act  best  in  watery  solution  as  against 
alcoholic  solutions.     Two  explanations  of  this  are  offered  : 

(1)  That  the  salts,  etc.,  in  water  dissociate  into  their  ions, 
and  the  latter  are  more  active  in  producing  chemical 
change ;    in   alcohols,   dissociation   does   not    take   place. 

(2)  That  the  alcohol  hinders  the  action  of  the  disinfectant 
by  hardening  the  bacterial  cell  wall.  In  favour  of  the 
second  reason  it  has  been  noted  that  while  absolute 
alcohol  is  useless  as  a  germicide,  added  to  aqueous  mercuric 
chloride  solution  in  the  proportion  of  25  per  cent  it  increases 
the  efficiency  of  the  disinfectant.  The  addition  of  NaCl, 
on  the  other  hand,  diminishes  the  efficiency,  it  is  believed 
by  reducing  the  number  of  free  ions. 

The  halogens  (CI,  Br,  I)  are  efficient  in  the  order  given. 
Chloride  of  lime  liberates  free  CI  when  treated  with  an 


378         PUBLIC     HEALTH    BACTERIOLOGY 

acid.  When  used  simply  in  solution,  oxygen  is  liberated 
in  the  nascent  state,  and  oxidizes  any  organic  matter 
present  (CaOCl2  +  H20  =  CaO  +  2HCI  +  O).  Peroxide 
of  hydrogen  and  potassium  permanganate  act  similarly 
as  oxidizers. 

Carbolic  acid  (phenol)  and  the  cresols  (lysol  and  creolin) 
do  not  dissociate,  and  their  efficiency  is  increased  by  the 
addition  of  NaCl  and  diminished  by  alcohol.  Formalde- 
hyde is  not  helped  by  adding  salt,  but  alcohol  is  harmful. 

Standardization.  —  The  emciency  of  any  disinfectant 
depends  on  many  factors,  namely,  the  strength  used,  the 
solvent,  the  temperature,  the  bacterium  to  which  applied, 
the  time  allowed  for  action,  the  other  substances  present, 
the  number  of  bacteria  present.  For  practical  purposes 
the  strengths  are  expressed  as  percentages  ;  but  in  com- 
parisons it  is  more  scientific  to  work  with  solutions  of 
the  molecular  weight  or  multiples  thereof  in  one  litre. 

Coefficient  of  Inhibition.  —  This  term  is  applied 
to  the  strength  (of  a  chemical  substance)  which  is  able  to 
prevent  the  growth  of  a  micro-organism  ;  that  is,  its 
antiseptic  value. 

It  is  determined  by  making  broths  or  other  media 
containing  the  chemical  substance  to  be  tested,  in  a  range 
of  strengths.  Equal  quantities  of  the  bacterium  used  in 
the  test  are  inoculated  into  the  various  tubes  (say,  one 
loopful),  the  contents  mixed,  and  incubated.  In  the 
case  of  solid  media,  they  are  poured  into  plates  and  the 
colonies  (if  any)  counted.  In  broth  cultures,  look  for 
turbidity,  and  confirm  positive  or  negative  results  by 
making  films.  The  coefficient  is  expressed  in  terms  of 
strength  and  bacterium  used.  Thus,  carbolic  acid  is 
said  to  inhibit  the  growth  of  anthrax  bacilli  when  present 
in  a  strength  of  1  part  in  800  of  medium.  B.  typhosus 
requires  1-400,  and  Sp.  cholerae,  1-600.  Of  corrosive 
sublimate,  1-100,000  inhibits  B.  anthracis,  and  1-60,000 
inhibits  B.  typhosus. 

Germicidal,  Bactericidal,  or  Disinfectant  Strengths. 

— Koch  used  anthrax  spores  dried  on  silk  threads,  which 
he  immersed  in  various  strengths  of  substance  being 
tested,   at  a  definite  temperature  and  for  varying  times. 


SPECIAL    EXAMINATIONS  379 

The  threads  were  then  removed  and  carefully  washed  in 
sterile  water  to  discharge  the  disinfectant.  They  were 
then  laid  on  gelatin  and  incubated,  and  the  result 
was  noted.  This  method  is  faulty,  in  that  it  is  difficult 
to  remove  the  disinfectant,  some  of  which  clinging  to  the 
bacteria  inhibits  growth.  The  Rideal-Walker  method 
may  be  applied  here  to  determine  germicidal  strength 
without  comparison  with  carbolic  acid. 

The  results  are  stated  in  terms  of  strength  used,  time 
exposed,  and  bacterium  killed :  thus  carbolic  acid  is 
bactericidal  to  anthrax  spores  at  ordinary  temperatures 
in  1-20  dilution  in  four  to  forty-five  days  ;  at  40°C,  in 
three  hours. 

To  Sp.  cholerae  at  ordinary  temperatures,  1-200  is 
fatal  in  five  minutes,  and  1-300  is  fatal  in  two  to 
twenty-four  hours. 

To  B.  typhosus  at  ordinary  temperatures,  1-50  is  fatal 
in  five  minutes. 

To  staphylococci  and  streptococci  at  ordinary  tempera- 
tures, 1-60  is  fatal  in  five  minutes. 

Corrosive  sublimate  is  fatal  to  anthrax  spores  in 
1-2000  in  twenty-six  hours. 

To  anthrax  and  typhoid  bacilli  and  Sp.  cholerae,  in 
1-2000  in  five  minutes. 

To  staphylococci  and  streptoccoci,  1-10,000  to  1-2000 
in  five  minutes. 

These  tests  supply  useful  data,  but  cannot  be  taken  as 
applying  to  the  action  of  the  same  disinfectants,  when 
mixed  with  the  body  fluids. 

Rideal-Walker  Test. — In  this  test  carbolic  acid  is 
taken  as  the  standard  disinfectant,  and  the  results  of  tests 
of  other  disinfectants  are  expressed  in  terms  of  their  power, 
compared  to  the  standard,  of  inhibiting  growth  of  the  same 
organism  in  the  same  time.  This  is  called  the  "  carbolic 
acid  coefficient  "  of  the  particular  disinfectant. 

Process.— A  series  of  accurate  dilutions  of  pure  carbolic 
acid  and  of  the  disinfectant  are  prepared  in  sterile  distilled 
water.  A  twenty-four  hours'  culture  at  370  C.  in  Lemco 
broth  (reaction  +  i*5)  of  B.  typhosus  is  used  as  the  test 
organism.  A  standard  size  of  platinum  loop  is  used  to 
make    the    subcultures.     The    disinfectant    solutions    are 


380         PUBLIC    HEALTH    BACTERIOLOGY 

arranged  in  a  series  of  tubes,  each  containing  5  ex.,  and 
the  dilution  of  the  disinfectant  is  marked  on  each  tube. 

To  5  c.c.  of  a  particular  dilution,  five  drops  of  the  filtered 
culture  are  added  ;  the  tube  is  shaken  and  set  down. 
Now  repeat  with  the  next  dilution,  and  so  on.  At  the 
end  of  2 -5  minutes,  a  subculture  is  made  from  the  tube 
first  inoculated,  into  5  c.c.  of  sterile  broth,  and  similarly 
with  all  the  dilutions,  in  the  proper  order.  At  the  end  of 
5,  7-5  10,  12-5,  and  15  minutes,  put  up  further  subcultures 
thus,  making  six  series  of  subcultures  in  all  from  each  tube. 
The  same  process  is  carried  out,  at  the  same  time,  with  the 
same  culture,  with  dilutions  of  carbolic  acid.  All  the  sub- 
cultures are  incubated  for  at  least  forty-eight  hours  at 
370  C,  and  the  presence  or  absence  of  growth  is  noted. 
From  the  table  of  results,  the  two  dilutions  doing  the  same 
work  in  the  same  time  are  seen.  Say  that  1-500  of  Disin- 
fectant A  inhibited  growth  in  2-5,  5,  7-5  minutes'  exposures, 
and  that  carbolic  acid  1-110  did  the  same  ;  then  the 
carbolic  acid  coefficient  of  Disinfectant  A  is  500  -=-  no  —  4-5. 

Since  its  introduction  this  test  has  been  much  used.  It 
has  also  been  subjected  to  much  criticism.  It  deals  with 
"  naked  "  bacteria.  The  other  objections  are  that  it  must 
be  carefully  done,  all  the  dilutions  should  be  accurate,  the 
organism  used  should  be  the  same  throughout,  etc.  These 
are  not  indictments  of  the  test  in  careful  hands,  and  where 
the  tests  on  different  disinfectants  are  made  by  the  same 
individual.  Still  the  results  obtained  by  this  test  should 
not  be  taken  for  more  than  they  pretend  to  be,  the  relative 
power  of  different  bodies  to  carbolic  acid  under  the  same 
conditions.  The  application  of  such  results  to  ordinary 
purposes  must  be  made  very  cautiously. 

The  "Lancet"  Commission  Test. — This  is  a  modified 
Rideal-Walker  test,  in  which  the  number  of  dilutions  is 
increased  up  to  nine,  and  the  time  periods  correspondingly 
up  to  thirty  minutes  ;  B.  coli  is  used  as  the  test  organism  ; 
MacConkey's  bile-salt  litmus  glucose  peptone  water  is  used 
as  the  medium  for  subcultures  instead  of  broth  ;  platinum 
spoons  are  used  instead  of  loops,  and  the  method  of  calcula- 
tion is  different. 

The  B.  coli  used  is  cultivated  for  twenty-four  hours  at 


SPECIAL    EXAMINATIONS  381 

370  C.  in  a  broth  made  by  mincing  i  lb.  of  fat-free  bullock's- 
heart  meat ;  macerate  it  in  cold  water  for  two  to  three 
hours ;  cook  over  a  small  gas  flame  for  two  to  three 
hours  more  ;  boil ;  filter ;  make  up  to  a  litre  ;  add  10  grm. 
each  of  NaCl  and  Witte's  peptones ;  and  standardize  to  an 
acidity  of  1-5  per  cent  to  phenolphthalein.  To  obtain  an 
emulsion  from  the  culture,  it  is  well  shaken  and  then 
filtered  through  a  double  layer  of  Swedish  filter-paper. 
The  carbolic  acid  dilutions  were  made  at  first  by  taking 
ac.  carbolic  B.P.  no  grm.  =  100  grm.  pure  phenol.  Later, 
dry  crystalline  carbolic  acid  was  taken  as  100  per  cent 
phenol.  The  dilutions  were  always  freshly  made,  as  it  was 
found  that  otherwise  they  lost  some  of  their  germicidal 
power,  even  when  kept  corked  and  in  the  dark.  The  dis- 
infectant dilutions  are  made  with  distilled  sterile  water,  and 
5  c.c.  of  each  are  put  in  small  glass  specimen  pots,  2-5  in. 
X  f  in.,  arranged  in  holes  on  a  board.  These  pots  are 
left  uncovered  during  the  experiment,  and  the  results  are 
not  vitiated  owing  to  the  MacConkey's  media  used  for  the 
subculturing,  inhibiting  most  other  organisms  than  B.  coli. 
Owing  to  the  platinum  spoon  holding  three  times  the 
amount  in  a  standard  loopful,  10  c.c.  are  used  for  the 
subculture  medium.  To  assist  in  rapid  work  a  special 
wheel  has  been  devised,  to  hold  the  spoons,  when  not  in 
use,  in  a  sterilizing  flame. 

Process. — All  the  apparatus  being  ready  and  to  hand, 
the  dilutions  of  the  unknown  disinfectant  are  "  seeded," 
each  getting  a  spoonful  of  the  B.  coli  emulsion,  and  being 
well  stirred.  The  seeding  is  begun  at  the  strongest 
dilution  and  proceeds  to  the  weakest.  At  the  end  of 
2-5  minutes,  a  spoonful  is  removed  from  the  first  dilution 
seeded  and  added  to  a  tubeful  (10  c.c.)  of  MacConkey's 
broth,  properly  labelled.  The  same  process  is  repeated 
with  all  the  tubes,  and  is  all  gone  over  again  after  5,  7-5, 
10,  12-5,  15,  20,  25,  and  30  minutes. 

The  same  procedure  is  followed  with  dilutions  of  carbolic 
acid. 

The  subcultures  are  incubated  for  forty-eight  hours  at 
370  C.  A  positive  result  is  indicated  by  the  medium 
turning  red,  and  the  formation  of  bubbles  of  gas.  In 
some  tubes  this  change  will  appear  in  twelve  to  fourteen 


382         PUBLIC    HEALTH     BACTERIOLOGY 

hours  as  a  purple  tinge,  becoming  pink  and  then  red  in 
another  two  hours.  The  results  are  filled  into  a  tabular 
form,  with  a  plus  sign  for  growth  and  a  zero  for  no  growth. 
The  coefficient  is  thus  calculated  :  The  weakest  dilution 
of  the  sample  under  test,  giving  no  growth  at  2-5  minutes, 
is  divided  by  the  weakest  dilution  of  carbolic  acting 
similarly  in  the  same  time  ;  the  weakest  dilution  giving 
no  growth  at  thirty  minutes  is  divided  by  the  same  for 
carbolic  acid ;  the  two  results  are  averaged,  and  the 
average  or  mean  is  taken  as  the  carbolic  acid  coefficient. 
Thus,  if  Disinfectant  B  gives  no  growth  with  1-220  in 
2 -5  minutes  and  with  1-340  in  30  minutes,  and  if  the 
dilutions  for  carbolic  acid  are  1-110  and  1-180,  then 
the  coefficient  would  be  : — 

(H»  +  Hi)  -  2  or  (2  +  i-88)  +  2  =  1-94. 

The  Commission  prefer  to  express  their  dilutions  as 
percentages,  and  when  this  is  done  the  mode  of  division 
is  reversed  ;  that  is,  the  weakest  percentage  (of  carbolic,  etc.) 
is  divided  by  the  weakest  percentage  of  the  test  substance, 
etc.  The  above  dilutions  become  for  B,  0-454  per  cent 
and  0-294  per  cent,  and  for  carbolic,  0-909  per  cent  and 
0-555  per  cent  respectively.  The  coefficient  therefore  is 
(%m  +  %'5U)  -  2  or  (2  +  1-85)  4-  2  *»  1-92.  The  tem- 
perature of  the  room  averaged  about  620  to  670  F. 

By  this  method  the  coefficients  ranged  from  0-025  to 
9-8  for  the  usual  coal-tar  disinfectants  on  the  market. 
Applied  to  corrosive  sublimate,  the  coefficient  at  2-5 
minutes  was  about  2000,  and  at  30  minutes  about  6000, 
giving  a  mean  of  about  4000.  Chloride  of  lime  similarly 
gave  figures  of  45  and  93,  or  a  mean  of  69.  For  formalin 
the  coefficient  was  o-6. 

Among  the  conclusions  reached  were  the  following  : 
That  results  obtained  in  such  bacteriological  experiments, 
although  giving  a  germicidal  value  to  a  disinfectant  under 
the  most  favourable  conditions,  afford  little  indication  of 
their  germicidal  value  when  used  in  practical  disinfection. 
That  much  remains  to  be  done  in  the  solution  of  such 
problems  (amongst  others)  as  arise,  due  to :  (1)  The 
presence  of  foreign  substances  in  the  material  to  be  dis- 
infected ;    (2)  The  temperature  at  which  the  disinfecting 


SPECIAL    EXAMINATIONS  383 

process  is  carried  on  ;  (3)  The  kind  of  water  used  for 
dilution — hard  water,  soft  water,  or  sea  water  ;  (4)  The 
type  of  micro-organism  that  has  to  be  dealt  with  ;  (5)  The 
nature  of  the  substance  to  be  disinfected  and  the  character 
of  its  surface  ;  (6)  The  time  to  be  allowed  for  the  process. 
That  only  when  the  influence  of  such  factors  can  be 
calculated  will  it  be  possible  to  modify  any  standard  co- 
efficient figure,  and  thus  to  obtain  data  for  the  preparation 
of  effective  and  economical  dilutions  for  the  practical 
problem  of  disinfection. 

The  inquiry  has  shown  that,  so  far  as  emulsions  are 
concerned,  those  which  contain  the  highest  quantity  of 
phenoloids  in  the  finest  state  of  division  and  having  the 
least  tendency  to  combine  with  albumins,  lime,  or  other 
foreign  substances  in  solution  (and  remain  combined),  will 
be  found  to  be  the  most  efficient  disinfectants. 

The  remarkable  parallelism  between  the  results  of  this 
inquiry  (as  shown  in  the  figures  for  the  coefficient)  and  the 
results  of  the  independent  chemical  one,  is  very  striking. 
The  chemical  commissioners  discovered  that  if  they 
subtracted  the  carbolic  acid  equivalent  of  the  bromine 
absorbed  by  the  percentage  of  phenoloids  present,  from 
the  percentage  of  phenoloids  present,  and  divided  the 
difference  by  3,  the  figures  obtained  in  many  instances 
are  the  same  (or  very  nearly  so)  as  those  assigned  as  the 
carbolic  acid  co-efficient  for  the  same  substance  by  the 
bacteriological  commissioners.  In  the  exceptions  the  dis- 
infecting fluids  did  not  form  an  emulsion  with  water,  nor 
show  Brownian  movements.     (See  p.  142.) 

^*  =  C.C. 

3 

Tables  showing  the  results  of  the  examination  (on  these 
principles)  of  the  disinfectants  in  common  use,  are  given 
in  the  Lancet  for  1909,  vol.  ii. 


APPENDIX. 

REGULATIONS     FOR     THE     DIPLOMA     IN 
PUBLIC     HEALTH. 

I. — The  Council,  having  regard  to  the  terms  of  Section  18 
of  the  Local  Government  Act  (1888)  and  of  Section  54  of  the 
Local  Government  (Scotland)  Act  (1889),  and  observing  that 
under  those  sections  special  privilege  is  to  be  accorded  to  the 
holders  of  the  diplomas  granted  under  Section  21  of  the 
Medical  Act  (1886),  and  therein  described  as  Diplomas  in 
Sanitary  Science,  Public  Health,  or  State  Medicine,  thinks  it 
essential  to  declare,  with  regard  to  its  own  future  action 
under  Section  21  of  the  Medical  Act  (1886),  that  it  will  not 
consider  diplomas  to  "  deserve  recognition  in  the  Medical 
Register  "  unless  they  have  been  granted  under  such  conditions 
of  education  and  examination  as  to  ensure  (in  the  judgment  of 
the  Council)  the  possession  of  a  distinctively  high  proficiency, 
scientific  and  practical,  in  all  the  branches  of  study  which 
concern  the  public  health  ;  and  the  Council,  in  forming  its 
judgment  on  such  conditions  of  education  and  examination, 
will  expect  the  following  rules  to  have  been  observed  : — 

Rule  i.  The  curriculum  for  a  Diploma  in  Sanitary  Science, 
Public  Health,  or  State  Medicine  shall  extend  over  a  period 
of  not  less  than  nine  calendar  months. 

Rule  2.  Every  candidate  for  a  Diploma  in  Sanitary  Science, 
Public  Health,  or  State  Medicine  shall  have  produced  satis- 
factory evidence  that,  after  obtaining  a  registrable  qualification, 
which  should  be  registered  before  admission  to  examination 
for  the  diploma,  he  has  received  practical  instruction  in  a 
laboratory  or  laboratories,  British  or  foreign,  approved  by 
the  licensing  body  granting  the  diploma  in  which  Chemistry, 
Bacteriology,  and  the  Pathology  of  the  Diseases  of  Animals 
transmissible  to  Man  are  taught. 

Note. — The  laboratory  instruction  shall  cover  a  period  of 
not  less  than  four  calendar  months,  and  the  candidate  shall 
produce  evidence  that  he  has  worked  in  the  laboratory  for 
at  least  240  hours,  of  which  not  more  than  one-half  shall  be 


APPENDIX  385 

devoted  to  practical  chemistry.  The  laboratory  course 
should  be  so  arranged  as  to  lay  special  stress  on  practical 
work  which  bears  most  directly  on  the  duties  of  a  medical 
officer  of  health. 

Rule  3.  Every  Candidate  shall  have  produced  satisfactory 
evidence — 

Either  (1)  that,  after  obtaining  a  registrable  qualification,  he 
has  during  six  months  been  diligently  engaged  in  acquiring 
a  practical  knowledge  of  the  duties,  routine  and  special,  of 
public  health  administration,  under  the  personal  supervision 
of  (a)  In  England  and  Wales,  the  medical  officer  of  health  of 
a  county  or  of  a  single  or  combined  sanitary  district  having  a 
population  of  not  less  than  50,000,  or  a  medical  officer  of 
health  devoting  his  whole  time  to  public  health  work  ;  or 
(b)  In  Scotland,  a  medical  officer  of  health  of  a  county  or 
counties,  or  of  one  or  more  districts  having  a  population  of 
not  less  than  30,000  ;  or  (c)  In  Ireland,  a  medical  superin- 
tendent officer  of  health  of  a  district  or  districts  having  a 
population  of  not  less  than  30,000  ;  or  (d)  In  the  British 
dominions  outside  the  United  Kingdom,  a  medical  officer  of 
health  of  a  sanitary  district  having  a  population  of  not  less 
than  30,000,  who  himself  holds  a  registrable  Diploma  in  Public 
Health  ;  or  (e)  A  medical  officer  of  health  who  is  also  a  teacher 
in  the  department  of  public  health  of  a  recognized  medical 
school  ;  or  (/)  A  sanitary  staff  officer  of  the  Royal  Army 
Medical  Corps  having  charge  of  an  army  corps,  district,  com- 
mand, or  division,  recognized  for  this  purpose  by  the  General 
Medical  Council  ;  or  (g)  An  assistant  medical  officer  of  health 
of  a  county  or  of  a  single  sanitary  district  having  a  population 
of  not  less  than  50,000,  provided  the  medical  officer  of  health 
of  the  county  or  district  in  question  permits  the  assistant 
officer  to  give  the  necessary  instruction  and  to  issue  certificates  ; 

Or  (2)  That  he  has  himself  held  for  a  period  of  not  less  than 
three  years  an  appointment  as  medical  officer  of  health  of  a 
sanitary  district  within  the  British  Dominions,  and  having  a 
population  of  not  less  than  15,000. 

Note  1. — The  certificate  for  the  purpose  of  Rule  3(1)  must 
include  testimony  that  the  candidate  has  attended  under  the 
supervision  of  the  person  certifying  on  not  less  than  60 
working  days.  Provided  that  if  the  candidate  has  : — (i)  Pro- 
duced satisfactory  evidence  that  he  has  attended  a  course  or 
courses  of  instruction  in  sanitary  law,  vital  statistics, 
epidemiology,  school  hygiene,  and  other  subjects  bearing  on 
public  health  administration,  given  by  a  teacher  or  teachers 

25 


386         PUBLIC    HEALTH    BACTERIOLOGY 

in  the  department  of  public  health  of  a  recognized  medical 
school ;  or  (ii)  Produced  evidence  that  he  has  been  a  resident 
medical  officer  in  a  hospital  for  infectious  diseases  containing 
not  less  than  ioo  beds,  during  a  period  of  three  months — the 
period  during  which  he  has  been  engaged  in  acquiring 
practical  knowledge  of  his  duties  under  this  rule  may  be 
reduced  to  three  months,  to  include  an  attendance  on  at  least 
30  working  days. 

Note  2. — For  the  districts,  commands,  and  divisions  that 
have  been  recognized  by  the  Council  under  Rule  3  (1)  (/)  see 
below. 

Rule  4.  Every  candidate  shall  have  produced  evidence  that, 
after  obtaining  a  registrable  qualification,  he  has  attended 
during  three  months  at  least  twice  weekly  the  practice  of  a 
hospital  for  infectious  diseases  at  which  candidate  has  received 
instruction  in  the  methods  of  administration. 

Note  1. — Methods  of  administration  shall  include  the 
methods  of  dealing  with  patients  at  their  admission  and 
discharge,  as  well  as  in  the  wards,  and  the  medical  super- 
intendence of  the  hospital  generally. 

Note  2. — In  the  case  of  a  medical  officer  of  the  Royal 
Army  Medical  Corps  a  certificate  from  a  principal  medical 
officer  under  whom  he  has  served,  stating  that  he  has  during 
a  period  of  at  least  three  months  been  diligently  engaged  in 
acquiring  a  practical  knowledge  of  hospital  administration 
in  relation  to  infectious  diseases,  may  be  accepted  as  evidence 
under  Rule  4. 

*„<*  The  Rules  2,  3,  4,  as  to  study,  shall  not  apply  to  medical 
practitioners  registered,  or  entitled  to  be  registered,  on  or 
before  Jan.  1st,  1890. 

Rule  5.  The  examination  shall  have  been  conducted  by 
examiners  specially  qualified  ;  it  shall  have  extended  over 
not  less  than  four  days,  one  of  which  shall  have  been  devoted 
to  practical  work  in  a  laboratory,  and  one  to  practical  examina- 
tion in,  and  reporting  on,  subjects  which  fall  within  the  duties 
of  a  medical  officer  of  health,  including  those  of  a  school 
medical  officer. 

II. — The  Council  shall,  from  time  to  time,  appoint  an 
inspector  or  inspectors  of  examinations  in  public  health,  with 
special  instructions  to  report  to  the  Council  whether  the 
examination  of  each  licensing  body  does  or  does  not  afford 
evidence,  on  the  part  of  candidates  passing  such  examination, 


APPENDIX 


387 


of  a  distinctly  high  proficiency,  scientific  and  practical,  in 
each  and  all  of  the  branches  of  study  which  concern  the 
public  health. 

List  of  the  districts  and  commands  that  have  been  recognized 
by  the  Council  under  Rule  3  (/)  : — 


Aldershot. 
Salisbury  Plain. 
Southern  and  South-Eastern 
Western. 

Dublin  and  Belfast. 
Cork. 

Chatham  and  Woolwich. 
Home. 
Eastern. 

North-Eastern  and  North- 
western. 
Scottish. 

Gibraltar  Command. 
Malta  Command. 


The   following   Indian  Divi- 
sions, viz.  : — 
1st  (Peshawar). 
2nd  (Rawalpindi). 
3rd  (Lahore). 
5th  (Mhow). 
6th  (Poona). 
7th  (Meerut). 
8  th  (Lucknow). 
9th  (Secunderabad). 
Burma. 
Quetta 


388 


PRESERVATIVES    IN    MILK    AND    CREAM. 

The  Local  Government  Board,  England,  in  February,  191 2, 
in  the  exercise  of  its  powers  under  the  Public  Health  Acts, 
drafted  regulations  prohibiting  the  use  of  preservatives  in 
milk  and  denning  the  conditions  under  which  preservatives 
may  be  used  in  cream. 

PRESERVATIVES  IN  MILK. 
Article  III.  :  "  1.  No  person  shall  add,  or  order  or  permit 
any  other  person  to  add,  any  preservative  substance  to  milk 
intended  for  sale  for  human  consumption.  2.  No  person 
shall  sell,  or  expose  or  offer  for  sale,  or  have  in  his  possession 
for  the  purpose  of  sale,  any  milk  to  which  any  preservative 
substance  has  been  added  in  contravention  of  subdivision  (1) 
of  this  article."  The  expression  "  milk  "  includes  separated, 
skimmed,  condensed,  and  dried  milk  ;  but  as  the  traffic  in 
condensed  milk  would  be  seriously  impeded  if  the  use  of 
sugar  were  disallowed,  it  is  provided  that  "  neither  cane  nor 
beet  sugar  shall  be  regarded  as  a  preservative  or  a  thickening 
substance." 

PRESERVATIVES  IN  CREAM. 
"  I.  No  person  shall  add,  or  order  or  permit  any  other  person 
to  add,  (a)  any  thickening  substance  to  cream  or  preserved 
cream  ;  {b)  any  preservative  substance  to  cream  containing 
less  than  40  per  cent  by  weight  of  milk  fat  ;  (c)  to  cream 
containing  40  per  cent  or  more  by  weight  of  milk  fat  any 
preservative  substance  other  than  (i.)  boric  acid,  borax,  or  a 
mixture  of  these  preservative  substances,  or  (ii.)  hydrogen 
peroxide,  in  amount  not  exceeding  0-1  per  cent  by  weight, 
in  any  case  in  which  the  cream  is  intended  for  sale  for  human 
consumption.  2.  No  person  shall  sell,  or  expose  or  offer  for 
sale,  or  have  in  his  possession  for  the  purpose  of  sale,  any 
cream  to  which  any  thickening  substance  or  any  preservative 
substance  has  been  added  in  contravention  of  the  provisions 
of  subdivision  (1)  of  this  article."  Every  seller  of  preserved 
cream  will  be  required  in  every  invoice,  bill,  advertisement, 
trade  list,  or  other  document  which  is  used  in  connection  with 
the  sale  of  preserved  cream,  to  describe  the  article  as  (a)  pre- 
served cream  (boracised),  or  (b)  preserved  cream  (peroxidised), 
as  the  case  may  be.     (This  provision  will  come  into  force  on 


APPENDIX  389 

January  i,  191 3.)  Dealers  in  cream,  preserved  in  a  manner 
which  does  not  contravene  the  above  regulation,  will  be 
required,  by  means  of  labels  on  the  receptacles,  to  declare 
that  the  cream  is  preserved  and  to  state  the  name  of  the 
preservative.  In  this  matter  the  regulations  are  precise, 
laying  down  in  a  schedule  the  size  of  the  label,  which  varies 
according  to  the  capacity  of  the  receptacle,  and  prohibiting 
the  attachment  to  any  receptacle  of  a  label  bearing  a  trade 
description  which  would  be  likely  to  mislead  a  purchaser  as 
to  the  utility  of  the  preservative  substance.  In  tea  shops 
and  other  refreshment  rooms  the  cream  jugs  will  not  be 
required  to  be  labelled  if  in  each  room  a  conspicuous  notice  is 
affixed  indicating  that  the  cieam  supplied  is  preserved  cream, 
or  if  a  statement  to  that  effect  is  printed  on  the  bills  of  fare. 
The  regulations  are  made  under  the  Public  Health  (Regulations 
as  to  Food)  Act,  1907,  and  any  person  who  wilfully  neglects  to 
carry  out  the  regulations  is  liable  to  a  penalty  not  exceeding 
^100,  and  in  the  case  of  a  continuing  offence  to  a  furthei 
penalty  not  exceeding  £50  for  every  day  during  which  the 
offence  continues.  The  penalties  for  offences  against  the 
regulations  are  those  for  which  provision  is  made  by  Sub- 
section (3)  of  Section  1  of  the  Public  Health  Act,  1896,  but 
before  the  local  authority  institutes  proceedings  against  any 
person  they  will  be  required  to  afford  him  an  opportunity  of 
explaining  the  circumstances  in  which  any  irregularity  may 
have  occurred.  With  the  exception  of  the  provision  relating 
to  the  labelling  of  preserved  cream,  which  takes  effect  on  Jan. 
1st,  191 3,  the  regulations  take  effect  from  June  1st,  191 2. 


390 


BOVINE     AND     HUMAN     TYPES     OF 
TUBERCLE     BACILLI.* 

i.     Smith  Reaction. 

Theobald  Smith,  in  1896,  first  drew  attention  to  the 
existence  of  two  types  oi  tubercle  bacilli  in  mammals,  and  in 
1898  he  published  a  systematic  comparative  study  of  bacilli 
isolated  from  man  and  cattle,  and  pointed  out  the  differences 
between  the  two  types  as  summarised  on  page  277.  Pursuing 
the  research  further,  he  found  that  when  grown  in  slightly 
acid  glycerin  broth,  the  two  types  give  different  reaction 
curves. 

Glycerin  Broth. — The  human  type  caused  at  first  a  lessening 
of  the  acidity  of  the  medium  until  it  became  nearly  but  not 
quite  neutral  ;  thereafter  the  acidity  increased  until  it  again 
approached  or  slightly  exceeded  the  original  reaction. 

The  bovine  type  in  the  early  stages  of  its  growth  rendered 
the  medium  less  acid,  neutral,  or  even  alkaline  ;  and  then  it 
may  so  remain  or  become  acid  again,  but  never  up  to  the 
original  degree. 

The  British  Royal  Commission  for  this  test  also  used 
glycerin  litmus  milk  (milk  freed  from  cream,  plus  5  per  cent 
of  glycerin,  plus  5  per  cent  of  5  per  cent  watery  solution  of 
Merck's  purified  litmus).  They  found  that  all  human  viruses 
which  grew  vigorously  on  this  medium  at  first  caused  an 
increase  of  alkalinity,  and  later  acidity  and  clotting  ;  if  the 
growth  was  less  vigorous,  acidity  resulted  without  clotting. 
The  bovine  viruses  which  grew  vigorously  in  the  medium 
caused  acidity  finally,  but  never  clotted  the  milk  ;  the  poorly 
growing  viruses  left  the  milk  alkaline.  They  concluded  that 
the  reaction  curves  can  be  so  grouped  as  to  form  a  scale  of 
the  final  reactions  with  complete  gradations  from  one  type 
to  the  other. 

2.     Cultural  Characters  (p.  15). 

The  results  of  our  work  have  led  to  the  conclusion  that 
there  is  no  constant  qualitative  cultural  difference  between 

*  Abstracted  from  Vol.  V.  of  "  Collected  Studies  from  the  Research 
Laboratory,  Department  of  Health,  City  of  New  York,  1910,"  by  Park  and 
Krumwiede,  and  others. 


APPENDIX  391 

the  human  and  bovine  types  of  tubercle  bacilli.  Quantita- 
tively, and  with  respect  to  the  effect  of  glycerin,  however, 
there  is  a  marked  difference  in  the  great  majority  of  cultures, 
so  great  in  fact  that  almost  without  exception  the  type  can 
be  determined  from  cultures  alone.  This  difference  is  constant 
in  one  factor  only,  viz.,  amount  and  rapidity  of  growth  in 
early  cultures.  In  classifying  our  cultures  according  to  this 
characteristic,  we  can  broadly  say  that  all  bovine  types  of 
bacilli  are  dysgonic  (sparse  growth)  and  all  human  types  of 
bacilli  are  eugonic  (moderate  or  luxuriant  growth).  The 
question  remains,  What  is  the  best  medium  for  eliciting  this 
difference  ?  Any  medium  used  must  fulfil  certain  conditions, 
i.  It  must  be  especially  adapted  for  the  growth  of  the 
eugonic  viruses,  so  that  the  best  possible  growth  is  obtained. 

2.  It  would  be  preferable  if  the  growth  of  the  dysgonic 
virus  were  somewhat  retarded  on  the  medium.  This  will 
widen  the  gap  as  far  as  possible. 

3.  The  medium  must  be  nearly  uniform  in  its  results  ;  that 
is,  growth  should  not  fluctuate  with  the  different  batches  of 
medium  used. 

We  have  found  that  glycerin  egg  is  by  far  the  best  medium 
for  this  purpose.  .  .  .  Cobbett  (Royal  Commission)  made 
comparisons  of  the  two  types  of  organisms  on  both  serum  and 
glycerin  serum.  While  the  human  cultures  were  far  more 
vigorous  with  the  use  of  glycerin  serum,  the  bovine  cultures 
were  either  restrained,  or  if  aided  by  its  presence,  the  increase 
in  growth  was  slight.  This  difference  he  found  in  early 
cultures  to  be  of  diagnostic  value  in  the  separation  of  the 
two  types.  We  also  noticed  that  primary  cultures  of  the 
bovine  type  repeatedly  failed  on  glycerin  egg,  whereas  the 
primary  cultures  of  the  human  type  were  usually  markedly 
increased  in  luxuriance.  The  reading  of  Cobbett's  results 
led  us  to  adopt  glycerin  egg  as  the  basic  differential  medium. 

Giving  then  the  results  in  terms  of  glycerin,  the  following 
general  conclusions  may  be  drawn. 

(a).  All  cultures*  growing  luxuriantly  on  glycerin  egg  from 
the  start  are  of  the  human  type. 

(b).  All  cultures*  growing  sparsely,  or  even  not  at  all,  on 

*  No  direct  cultures  were  attempted.  Guinea-pigs  were  inoculated  and 
killed  in  three  to  five  weeks,  and  inoculations  made  from  tuberculous 
lymph  nodes  and  spleen  on  to  plain  egg  media  (Dorset),  glycerin  egg 
media  (Lubenau),  and  glycerin  potato.  Intravenous  inoculation  into 
rabbits  was  used  in  confirmation  of  type  of  culture.  If  rabbit  survived 
injection  of  1  mgr.  of  culture  for  60  days,  then  human  type. 


392 


PUBLIC     HEALTH    BACTERIOLOGY 


glycerin  egg  in  the  first  few  generations  are  of  the  bovine 
type. 

Glycerin  egg  (Lubenau).  Ten  eggs  are  blown  into  a  flask  and  200  c.c. 
of  5  per  cent  glycerin  bouillon  (neutral  or  moderately  alkaline)  added. 
The  further  preparation  is  as  for  plain  egg  medium. 

3.     Results. 
Table  of  Series    of   Non-selected  Cases    of   every  Type  of 
the  Disease,  showing  Type  of  Bacillus  Isolated. 


Adults 

Children 

Children 

Form  of  Tuberculosis 

16  years  and  over 

5-16  years 

0-5  years 

Human 

Bovine 

Human 

Bovine 

Human 

Bovine 

Pulmonary 

278 

— 

8 

5 

— 

Adenitis,  cervical 

9 

— 

19 

8 

6 

12 

Do.     inguinal    and 

axillary 

1 

— 

4 

— 





Abdominal 

1 

— 

1 

1 



3 

General  (alimentary  in 

origin) 

— 

— 

— 

— 

1 

1 

General 

2 

— 

1 

— 

12 

4 

♦General  +  Meningitis 

— 

— - 

— 

— 

18 

1 

Meningitis 

— 

— 

1 

— 

M 

1 

Bones  and  Joints 

1 



10 

— 

6 

— 

Genito-urinary 

3 

I 

1 

— 

— 

—  • 

Abscesses 

1 

435 

296 

1 

45 

9 

62 

22 

297 

54 

84 

4.   Conclusions  (Park  and  Krumwiede)  loc.  cit.  p.  134. 

Tubercle  bacilli  as  isolated  from  man  fall  into  two  groups. 
One  of  these  groups  is  identical  in  all  its  characters  with  that 
found  in  cattle.  That  is,  all  tubercle  bacilli  from  man  and 
cattle  fall  into  two  groups,  which  have  been  designated  the 
human  and  bovine  types. 

Each  type  shows  certain  differences,  the  most  important 
for  separation  being  those  culturally  and  in  virulence.  The 
great  majority  of  cultures  group  themselves  around  two 
extremes,  from  which  there  are  a  few  cultures  showing  variant 
characteristics.  There  is  no  overlapping  of  characteristics. 
The  two  types  are  probably  different  because  of  residence  in 
different  hosts  over  long  periods  of  time,  and  as  such  are 
stable.  The  evidence  in  favour  of  rapid  change  of  type  is 
incomplete  and  inconclusive. 


*  Includes  1  double  infection  :    Mesenteric  nodes   gave   human  type, 
meningeal  fluid  gave  bovine  type. 


393 


INDEX 

PAGE 

PAGE 

A  BIOGENESIS     . . 

I48 

Air,  bacteriological  examina- 

i\    Acarus  sacchari,  or  sugar 

tion  of 

363 

mite 

I09 

—  carbon  monoxide  in 

69 

Accelerated  reaction 

215 

—  estimation      of     C02     by 

Acetic  acid  in  vinegar 

124 

Haldane's  method     . . 

64 

Achorion  Schoenleinii 

343 

Hesse's  method    . . 

65 

Acid  in  beer,  spirit  indicatior 

L 

—  —  —  Lunge  and  Zecken- 

value  of 

145 

dorf's  method 

65 

—  permanganate    in    Tidy's 

—  —  —  Pettenkofer's  methoc 

63 

process 

44 

Scurfield's  apparatus 

66 

Acid-fast  bacilli  in  sections. 

167 

—  examination  of     . . 

62 

—  bacteria     . .          . .         163,  284 

—  ground 

7i 

Acidimetry  and  alkalimetry 

16 

—  moulds  v.  bacteria  in 

364 

Acidity  of  beer 

128 

—  noxious  emanations  in    . . 

67 

—  bread 

100 

—  organisms  found  in 

364 

Actinomyces 

286 

—  oxidizable      and      organic 

Actinomycosis 

286 

matter  in 

66 

Active  immunization 

184 

—  oxygen  in . . 

69 

Adams'  process  for  fat  in  milli 

:       75 

—  ozone  in     . . 

66 

Adonite 

106 

—  suspended  matter  in 

69 

Adulteration  of  butter 

87 

Albuminoid  ammonia  in  water 

43 

—  cereals 

97 

Alcohol  in  beer,  direct  distilla- 

— cocoa 

119 

tion 

128 

—  coffee 

113 

Tabarie's  method 

128 

—  flour 

99 

—  ethyl           

132 

—  honey 

in 

—  methyl 

134 

—  milk,  calculation  in 

76 

—  table,  short 

144 

—  mustard     . . 

112 

Alcohol-fast  bacteria 

163 

—  pepper 

112 

Aldehydes  in  spirits 

134 

—  tea 

ii5 

Alexines           . .          . .         183, 

203 

Aerated  bread 

100 

Alkalimetry  and  acidimetry 

16 

—  waters,  analysis  of 

53 

—  method     (Hehner's)     for 

Aerobic  sporing  bacilli 

304 

hardness  in  water 

37 

Afterdamp  in  mines  . . 

70 

—  sources  of  error  in 

17 

Agar,  beer-wort 

344 

Alkaline  permanganate 

43 

—  glucose 

153 

Alum  in  baking  powders     . . 

IOI 

—  glycerin 

153 

—  bread 

100 

—  lactose 

153 

—  flour 

99 

—  litmus  lactose 

153 

Aluminium  process  for  nitrates 

—  nutrient 

•      153 

in  water 

50 

—  rat 

•      255 

Amboceptor 

203 

—  salt 

•      255 

Ambulant  plague 

260 

Agglutination              . . 

.      191 

Ammonia  in  water,  albuminoid 

43 

—  in  cholera 

•      322 

—  —  free  and  saline 

40 

—  test  in  glanders    . . 

•      253 

—  chloride,  standard  solution 

—  in  typhoid  fever  . . 

234 

of          

4i 

Agglutinins 

192 

Amyl  alcohols 

134 

Aggressins       . .          . .         17 

7,  197 

Amylum  or  starch 

104 

Agin  B.  coli    . . 

•     357 

Anaerobic  sporing  bacilli 

304 

394 


INDEX 


PAGE 

Analyses  of  Glasgow  sewage 

and  effluents  : .  . .       61 

—  sewage  and  sewage  effluents, 

tables  of  . .  56,  6 1 


Autoclave  sterilization          . .  155 

Autolysins       ..          ..          ..  211 

Avian  tubercle  bacilli  278,  291 

Avogadro's  law          . .          . .  14 

•281 


—  of  waters,  table  of 

55 

Analysis  of  aerated  waters  . . 

53 

Bacillary  emulsion 

—  chemical    . . 

5 

Bacilli   of   the   haemorrhagic- 

—  colorimetric 

5 

septicaemic  group 

—  gravimetric 

5 

Bacillus  acidi  lactici  (Hueppe) 

—  of  ice 

53 

(Leichmann)        242 

—  of  mineral  waters 

53 

—  of  acute  conjunctivitis  . . 

—  of  sewage  from  midden  towns  56 

—  aerogenes  capsulatus 

—  volumetric 

5 

—  of  angular  conjunctivitis 

—  of  water     . . 

19 

—  anthracoides 

interpretation  of  results 

53 

—  anthracis    . . 

Anaphylaxis    . .          . .          180 

212 

—  of  Bordet-Gengou 

—  absolute     . . 

180 

—  botulinus 

—  acquired    . . 

218 

—  Bulgaricus 

—  classification 

218 

—  of  chicken  cholera 

—  to  serum-globulin 

217 

—  coli,  agin  . . 

—  white  of  egg 

215 

communis 

Aniline  oil-water  stains 

161 

typical 

Annatto  in  butter 

95 

fermenting  saccharose 

Anthrax  bacillus        . . 

304 

flaginac 

—  orders 

309 

isolation  of,  from  water 

Antianaphylaxis 

215 

sagin     . . 

—  to  white  of  egg 

217 

search  for,  in  water  . . 

Antibody-producer 

203 

typical    (British    Com- 

Antigen 

203 

mittee) 

—  by  complement-fixation . . 

210 

typical  (Houston) 

Antiplague  serum 

261 

—  diphtheria . . 

Antiseptic 

135 

—  of  Ducrey . . 

Antitoxic  sera 

189 

—  dysenteries  . . 

Antitoxin,  diphtheria 

248 

—  enteritidis  (Aertryck) 

Antituberculous  sera 

282 

(Gaertner) 

Appendix,  chemical  . . 

144 

sporogenes 

—  short  alcohol  table 

144 

—  faecalis  alcaligenes 

—  table  of  degrees  of   spirit 

—  fusiformis  . . 

indications 

146 

—  of  glanders 

—  —  Glaisher's  factors 

144 

—  Hoffmanni 

spirit  indication  value 

—  of  human  plague . . 

of  acetic  acid  in  beer 

145 

—  influenzas   . . 

weights    of    1    eft.    of 

—  of  Johne    . .          . .        28=5, 

water  vapour 

145 

—  of   Klebs-Loeffler . . 

Arabinose 

105 

—  of  Koch -Weeks    . . 

Arsenic  in  beer 

129 

—  lactis  aerogenes   . . 

Arsenious  solution,  standard 

135 

—  leprce 

Artificial  immunity 

184 

—  of  malignant  oedema 

Ascospores 

33i 

—  mallei 

Ash  of  milk 

74 

—  megatherium 

Asparagine 

106 

—  mucosus  capsulatus 

Aspergillosis 

336 

—  of   M orax-Axenfeld 

Aspergillus 

335 

—  mycoides   . . 

—  flavus 

336 

—  ozaenae 

—  glaucus 

335 

—  paratyphosus 

—  repens 

336 

—  pestis          . .          . .        '  . . 

—  fumigatus 

335 

—  phlegmonis      emphysema- 

Atomic  weights,  table  of 

13 

tosae 

319 


INDEX 


395 


PAGE 

PAGE 

Bacillus  pneumonia  . . 

240 

"  Black  death  "         . .         254, 

260 

—  of  quarter  evil 

318 

Black  rat 

258 

—  of  rabbit  septicaemia 

254 

Blastomycoses 

333 

—  radicosus   . . 

309 

Bleaching  of  flour 

99 

—  of  rhinoscleroma  . . 

242 

—  powder 

135 

—  of  septic  pleuropneumonia 

254 

Blood  films 

168 

—  smegmatis 

285 

■ —  serum,  Loe frier's  . . 

246 

—  of  soft  chancre     . . 

245 

medium 

154 

—  subtilis 

310 

"  Blowers  "     . . 

70 

—  of  swine  plague    . . 

254 

Boiled  milk,  detection  of 

79 

—  tetani 

311 

Borax  in  milk               80  and  errata 

—  tuberculosis 

273 

Bordet-Gengou  bacillus 

244 

—  typhosus     . . 

231 

Bordet  and  Gengou  reaction 

204 

—  typi  murium 

236 

Boric  acid  in  butter  . . 

92 

—  vulgatus    . . 

310 

milk 

80 

—  of  whooping  cough 

244 

Botulus  bacillus 

316 

—  xerosis 

247 

Bottom  yeast             . .         127, 

333 

—  of  Zur  Nedden 

245 

Bouillon  filtre 

281 

Baking  powders,  composition 

Bovine  tubercle  bacilli  277, 

289 

,390 

of          

IOI 

—  tuberculosis 

293 

definition  of   . . 

IOI 

Brandy 

132 

Bacterial  activity,  results  of 

172 

Braxy 

3i8 

—  poisons 

175 

Bread,  manufacture  of 

99 

—  protein 

175 

—  acidity  of 

100 

Bacteriological  examination  of 

—  aerated 

100 

air 

363 

Brewer's  yeast 

332 

dust 

366^ 

Brewing-waters 

127 

- —  —  milk 

Broth,  "  ghee  " 

256 

sewage,  etc.     . . 

365 

—  glycerin     . . 

390 

soil 

365 

—  nutrient 

I5i 

—  —  water    . .          . .          21, 

345 

■ —  standardization  of  its  re- 

 value  of    . . 

54 

action 

I5i 

—  media,  classified 

151 

Bromine    absorption    process 

Bacteriolysins 

191 

for  phenol       . .         141, 

137 

Bacterioscopic  examination  of 

Brown  rat 

258 

water 

347 

Brownian  movements  in  dis- 

Barley-sugar 

103 

infectants 

140 

Baryta  water             . .     10,  3s 

!,    6l 

Brucine   test   for   nitrates 

111 

Bates'  saccharometer 

126 

water 

49 

Bed  bug 

260 

Buboes  in  rats 

262 

Beer 

125 

Bubonic  plague 

257 

—  acidity  of              . .          128 

145 

Buchanan's  rat  agar  . . 

255 

—  addenda     . . 

I30 

Buchner's  tube 

3ii 

—  arsenic  in 

129 

Buddeized  milk 

83 

—  wort           . .          . .         126, 

128 

Budding 

331 

agar     

344 

Bug,  bed 

260 

Beers  from  starches,  etc. 

127 

Butter,  adulterations 

87 

Beeswax,  melting-point  of  . . 

in 

—  average  composition  of 

87 

Beet  sugar 

103 

—  bacteriology  of     . . 

376 

Benzoates  in  milk 

82 

—  boric  acid  in 

.92 

Bicarbonates  in  water,   with 

—  colouring  matter  in 

95 

carbonates 

31 

—  curd  or  casein  in  . . 

88 

with  free  C02 

32 

—  detection  of  starch  in 

94 

Bile-salt,  neutral-red,  lactose 

—  fat  in 

88 

agar 

154 

—  v .  margarine 

95 

—  glucose     litmus     peptone 

—  polariscope  test    . . 

94 

water 

153 

—  salt  in 

88 

Black  damp  in  mines 

70 

—  saponification  equivalent 

of 

93 

396 


INDEX 


Butter,  water  in 
Butter-fat,  detection  of  cotton 
seed  oil  in 

—  detection  of  sesame  oil  in 

—  fixed  fatty  acids  in 

—  iodine  absorption  of 

—  refractive  index  of 

—  sesame  oil  in 

—  specific  gravity  of 

—  valenta  test  for   . . 

—  volatile  fatty  acids  in 


PAGE 

88 


94 
94 
9i 
94 
92 
94 
92 
92 
88 


Cacao  butter  in  cocoa 

Cadaverin 

Caffeine  in  coffee 

—  tannate  in  tea  infusions 

—  in  tea 
Calculation  method  for  fat  in 

milk 
Calmette's  test 
Cane  sugar 
analysis  of 

—  —  in  milk 
Capsulated  bacilli 
Capsule  staining 
Carbol-fuchsin  (Ziehl-Neelsen) 
Carbol-glycerin-fuchsin 
Carbol-methylene-blue 
Carbolic    acid,    bromine    ab 

sorption  process       141, 
coefficient,  formula  for 

—  —  —  relation  to  per  cent 

of  phenoloids 
tests  for 

—  powders 
Carbohydrate  reactions  (short 

table  of) 
Carbohydrates,  classification  of 

—  definition  of 

—  table  of 
Carbon  dioxide  in  water,   as 

bicarbonate    and    car- 
bonate 

as  free  C02 

—  and  bicarbonate 

Carbon  monoxide  in  air 
Carbonates    in     water,     with 

bicarbonates 
Carbonic    acid    gas    in    air, 

estimation  of. .  63-66 

Casein  in  butter        . .  . .        88 

Cases  of  human  tuberculosis       294 
Centinormal   solution,    defini 

tion  of . . 
Ceratophyllus  unisus 
Cereals,  adulterations  in 

—  analysis  of 


117 
173 
113 
116 
116 

76 
281 
103 
106 

78 
240 
163 
161 
161 
161 

137 
143 

143 
137 
138 

240 
102 
102 
106 


31 
30 
32 
69 

31 


269 
97 
96 


Cereals,  animal  and  vegetable 

parasites  in     . . 
Cervical  buboes  in  rats 
Chancroid  bacillus     . . 
Charbon  symptomatique 
Cheese,  analysis 

—  average     composition     of 

Cheddar 

—  bacteriology  of     . . 

—  poisonous  metals  in  rind  of 

—  ripening  of 

—  varieties  of 
Chemical  analysis 

of  tubercle  bacilli 

water,  value  of 

—  examination  of  water 

—  properties     of     tubercle 

bacillus 

—  sterilization 
Chemiotaxis 
Chicory 

—  in  coffee 

China  ink  method     . . 
Chloride  of  tin  in  sugar 
Chlorides  in  water     . . 
Choke  damp  in  mines 
Cholera  carriers 

—  red  reaction 
Cider    . . 

—  vinegar 
Cimex  lectularius 
Citric  acid  in  lime  juice 
Citrus  limetta 

—  limonum 
Classification  of  fungi 
Clotted  cream 

C02  in  air,  Haldane's  method 

Hesse's  method 

Lunge  and  Zeckendorf 

method 

Pettenkofer's  method 

Scurfield's  apparatus 

Coal  dust  in  mines   . . 

—  tar  disinfectants  . . 
Cobbett  on  portals  of  entry 

of  tubercle  bacilli 

—  on  glycerin  media 
Cobra  venom 
Coca 
Cocoa   . . 

—  nibs 
Coco-nut  oil  in  butter 

estimation  of 

melting  point  of 

—  —  Reichert  figure  of 

source  of 

Cocos  nucifera 
Coffee 


262 

245 

318 

96 

96 

376 

96 

95 

95 

5 

274 

54 

,  22 

293 

155 

182 

113 

114 

328 

109 

24 

70 

322 

321 

132 

123 

260 

120 

120 

119 

170 

84 

64 

65 

65 
63 
66 
70 
140 

279 

391 

178 

119 

117 

117 

89 

91 

118 

90 

118 

118 

113 


INDEX 


397 


PAGE 

Coffee,  detection  of  chicory  in  114 

Colon  bacillus             . .          . .  230 

Colon-typhoid-dysentery  group  230 

Colorimetric  analysis             . .  5 

Colour  formation  by  bacteria  158 

—  reduction  by  bacteria      . .  159 
Colouring  matter  in  butter  . .  95 

sugar    . .          . .          . .  109 

wine      . .          . .          . .  131 

Columella         . .          . .          . .  334 

Comma  bacillus          . .          . .  321 

Committee   of   R.I.   of   P.H., 

report  of          . .          . .  347 

Complements  . .          . .         183,  203 

Condensed  milk          . .          . .  85 

—  skimmed  milks     . .          . .  85 

Coniferin          . .          . .          . .  106 

Copper  in  green  peas            ..  112 

—  sulphate     . .          . .          . .  139 

—  —  in  bread          . .          . .  100 

—  in    water,     estimation    of 

amount             . .          . .  28 

—  —  presence  of                 . .  25 
Copper-zinc     couple     process 

for  nitrates  in  water . .  51 

Corn  cockle  in  flour  . .          . .  99 

Cornish  cream             . .          . .  84 

Cotton-seed  oil  in  butter  fat  94 
Cow's     milk,     average     com- 
position of      . .             73,  86 

Cow-wheat  in  flour    . .          . .  99 

Cream,  composition  of          . .  84 

—  fat  in          . .          . .          . .  84 

—  in  milk       . .          . .          . .  73 

—  preservatives  in,  new  regu- 

lations..          ..          ..  388 

—  of  tartar  in  wine. .          . .  130 

Cresols..          ..          ..          ..  138 

Crith,  the        . .          . .          . .  14 

Cultivated  yeasts       . .          . .  333 

Cultural  methods       . .          . .  155 

—  reactions   . .          . .          . .  156 

Curd  in  butter            . .          . .  88 

Cutaneous  plague      . .          . .  260 

—  test  with  tuberculin        . .  280 
Cytases            . .          . .         183,  203 

Dalmarnock     sewage     and 

effluent  . .  . .  61 
Dalmuir  sewage  and  effluent  61 
Danysz's  virus  . .  . .  237 
Darnel  seeds  in  flour  . .  99 
Dauglish's  system  of  bread- 
making  . .  . .  100 
Decimal  mode  of  dilution  . .  367 
Decinormal  solution,  defini- 
tion of  . .  . .  8 
Demerara  sugar         . .          . .  109 


PAGE 

Denys  bouillon  nitre             . .  281 

Deodorant       . .    .       . .          . .  135 

Deviation  of  the  complement  211 

Devonshire  cream      . .          . .  84 

Dextrin            . .          . .          . .  105 

Dextrose          . .          . .          . .  104 

Dhobie's  itch . .          . .          . .  338 

Diagnosis  of  anthrax            . .  307 

—  plague    ...          . .          . .  262 

in  China           . .          . .  269 

Differentiation  of  B.  typhosus 

from  B.  coli    . .          . .  238 

—  staphylococci        . .          . .  222 

—  streptococci           . .          . .  223 
Diphenylamine    test    for    ni- 
trates in  water           . .  50 

Diphtheria  antitoxin             . .  248 

—  bacillus      . .          . .          . .  246 

Diplococcus  pneumoniae        . .  224 
Diploma    in    Public    Health, 

regulations      . .          . .  384 

Dirt  in  milk    . .          . .          . .  84 

Disaccharids  or  sugars          . .  102 

Discontinuous  sterilization  . .  155 

Disinfectants  . .          . .          . .  135 

—  bacteriological     examina- 

tion of              . .          . .  377 
standardization  of     . .  378 

—  chemical  examination  of  135 

—  co-efficient  of,  inhibition  of  378 

—  germicidal  strengths  of  . .  378 

—  Lancet  Commission  test  . .  380 

—  Rideal-Walker  test          . .  379 

—  standardization  of,  chemical  139 

—  in  watery  solutions         . .  377 
Disinfection     . .          . .          . .  155 

Disr/osal    of    dead    in    plague 

in  China          . .          . .  271 

Dorset's  egg  medium            . .  275 

Dried  milk      . .          . .          . .  83 

Dried  mother's  milk,  compo- 
sition of           . .          . .  86 

Drigalski     and     Conradi's 

medium            . .          . .  239 

Dry  heat  sterilization           . .  155 

—  wine            . .          .  •          •  •  130 
Ducrey's  bacillus       . .          . .  245 
Dulcite             . .          .  •          . .  105 
Dust,    bacteriological    exam- 
ination of        . .          . .  365 

Dysentery  bacillus    . .          . .  237 

Earth  bacillus           . .          . .  310 

Eberth's  bacillus        . .          . .  231 

Egg  medium,  glycerin           . .  391 

—  —  Dorset's           . .          . .  275 
Ehrlich's  side-chain  theory..  202 

—  theory  of  immunity        . .  199 


398 


INDEX 


Endo-enzyme 

332 

Endo's  medium 

239 

Endotoxins 

176 

Enrichment  method  of 

HofY- 

man  and  Ficker 

361 

Enzymes 

332 

Ergot  in  flour 

100 

Erythrasma 

338 

Escherich's  B.  coli    . .  ' 

230 

Ethers,  compound     . . 

133 

Ethyl  alcohol 

132 

Examination  of  milk 

73 

—  pus. . 

224 

Excretion  of  tubercle 

bacilli 

in  milk 

298 

External  anthrax 

304, 

306 

Extracellular  toxins . . 

176 

Farcy..           ..          ..          ..  251 

—  buds           . .          . .          . .  251 

—  order  of  1907        . .          . .  253 
Fat  in  butter,  Polenske  pro- 
cess      . .          . .          . .  90 

Reichert-Wollny     pro- 
cess         88 

—  cocoa          . .          . .          . .  117 

—  coffee          . .          . .          . .  114 

—  milk,  Adams'  process     . .  75 
calculation  method   .  .  76 

—  —  Gerber's  process         . .  75 

Leffmann-Beam  process     75 

maceration  process   . .  76 

Werner-Schmidt  method    74 

Fate    of    tubercle    bacilli    in 

tissues              . .          . .  297 

Favus ,  343 

Feeding     experiments     with 

tubercle  bacilli           . .  297 
Fehling's    solution :     compo- 
sition of          . .          . .  78 

Pavy's  modification  of  97 

Ferrous  sulphate       . .          . .  137 
Film,  to  make  a        . .          . .  161 
Filtration  of  immune  serum  204 
Final  report  of   Royal  Com- 
mission    on     tubercu- 
losis        288 

Findlay  on  portals  of  entry 

of  tubercle  bacilli       . .  279 

Finings             . .          . .          . .  127 

Fire-damp  in  mines  . .          . .  70 

Fixateur  . .  . .         199,  203 

Fixation  of  blood  films         . .  169 

—  the  complement  . .          . .  204 
Fixed  fatty  acids  in  butter- 
fat         . .          . .          . .  91 

Flagella  staining         . .          . .  165 

Flaginac  B.  coli         . .          . .  357 


PAGE 

Flaginac  group   of   reactions 

230,  356 

Fleischwasser . .           ..          ..  152 

Flexner  group             . .         • . .  238 

Flour,  ergot  in  . .  100 
Food    infection    with    avian 

tubercle  bacilli  . .  300 
bovine  tubercle  bacilli  301 

—  —  human  tubercle  bacilli  300 
Formaldehyde            ..          ..  136 

—  in  milk  . .  . .  80,  81 
Formalin  in  milk,  proportions 

used      . .          . .          . .  81 

—  quantitative  test  for  . .  136 
Formula     for     carbolic     acid 

co-efficient       . .          . .  143 
Forschammer     process     for 
organic  matter  in  water 

39,  44 

Frambcesia      . .          . .          . .  329 

Fraenkel's  pneumococcus  . .  224 
Frankland's    method    of    air 

examination  . .  . .  363 
to     estimate     organic 

matter  in  water          . .  38 

Free  mineral  acid,  tests  for..  121 

—  and    saline    ammonia    in 

water    . .          . .          . .  40 

Friedlaender's  bacillus          . .  240 

Fructose          . .          . .          . .  104 

Fruit  sugar     . .          . .          . .  104 

Fuchsin,  carbol-         . .          . .  161 

—  carbol -glycerin  ..  ..  161 
Fungi   . .          . .          . .          . .  170 

—  ringworm  . .          . .          . .  341 

Furfurol           . .          . .          . .  133 

Fusel  oil,  tests  f or     . .          . .  134 

Fusiform  bacillus       . .          . .  247 

Gaertner's  bacillus. .          ..  237 

Galactose         . .          . .          . .  104 

Gases  in  mines,  table  of       . .  70 

—  volume  and  density  of  . .  14 
Gelatin,  liquefaction  of        . .  159 

—  liquefying  organisms,  table 

of           . .          . .          . .  160 

—  nutrient     . .          . .          . .  152 

—  sugar  media,  Houston's..  358 
Gemmation     . .          . .          . .  331 

Gerber's    process    for    fat    in 

milk      . .          . .          . .  75 

Germicide        . .          . .          . .  139 

Ghee  broth     . .          . .          . .  256 

Giemsa's  stain            . .          . .  168 

Gin       . .          . .          . .          . .  132 

Ginger               ..          ..          ..  112 

Glaisher's  factors,  table  of  . .  144 

Glanders          ..          ..          ..  251 


INDEX 


399 


Glanders  bacillus 
—  order  of  1907 
Glasgow  sewage,  analyses  of 


103,  104, 


PAGE 
250 

253 
6l 

I09 
••  153 
••    152 

I07 

no 
III 
103,  104,  109 
rotatory 


Glucose 

—  agar 

—  broth 

—  in  cane  sugar 

—  golden  syrup 

—  honey 

—  starch  sugar 

—  syrup,     specific 

power  . .          . .          . .  no 

Glucoses  or  monosaccharids . .  102 

Glutin  in  flour           . .          . .  98 

Glycerin  agar..          ..          ..  153 

—  broth          . .          . .          . .  390 

—  egg  medium          . .          . .  391 

—  litmus  milk            . .          . .  390 

Glycogen          . .          . .          . .  105 

Golden  syrup..          ..          ..  no 

—  —  specific  rotatory  power  no 
Gonococcus     . .          . .          . .  227 

Gram's  method  for  sections. .  167 

of  staining       . .          . .  162 

Gram-negative     organisms, 

table  of  . .  . .  162 
Gram-positive     organisms, 

table  of           . .          . .  162 

Gravimetric  analysis             . .  5 

Granule  staining        . .          . .  166 

Grape  sugar    . .          . .          . .  104 

Greensands  strata,   effect  on 

water   . .  . .  47,  55 

Griess's    test    for   nitrites    in 

water    . .          . .          . .  47 

Ground  air                  . .          . .  71 

—  moisture    . .          . .          . .  72 

—  water          . .          . .          . .  72 

Grueber's  reaction     . .          . .  236 

Guarana           ..          ..          ..  119 

Guinea-pig  test  for  glanders  252 

H^molysin-producing     or- 
ganisms           . .          . .  161 
Haemolysis       . .          . .         160,  192 
Haffkine's  vaccine  for  plague  261 

—  —  for  cholera       . .          . .  323 
Haldane's  method  for  estima- 
tion of  C02  in  air       64,  69 

Han  ta            . .          . .          . .  264 

Hardness  in  water,  definition 

of          34 

determination     of    by 

standard  soap  solution  35 

—  —  estimation      of     by 

Hehner's  method       . .  37 

total,    temporary    and 

permanent       . .  37,  38 


Harrison's  indicator  in   Feh- 

ling's  test        . .          . .  108 

Hay  bacillus  ..          ..          ..  310 

Head  mould  . .          . .          . .  334 

Heavy  oils  in  carbolic  acid  . .  138 
Hehner's  alkalimetry  method 
to  estimate  hardness  in 

water    . .          . .          . .  37 

—  and  Richmond's  formula  76 

—  test   for   formaldehyde  in 

milk      . .          . .          . .  80 

Hesse's  method  of  air  exam- 
ination. .          . .          . .  363 

Heterolysins    ..          ..          ..  211 

High  fermentation     . .          . .  333 

High-pressure  sterilization  . .  155 

Histricopsylla  genus              . .  269 

Hoffmann's  bacillus  . .          . .  247 

Hoffman  and  Ficker's  medium  239 

Homogenized  milk     . .          . .  83 

Honey..           ..          ..          ..  Ill 

—  adulteration  of     . .          ..  in 

—  specific  rotatory  power  of  111 
"  Honey "  growth     . .          . .  251 

Honeydew       ..          ..          ..  Ill 

Houston's  lemco  medium     . .  222 

—  method  for   water   exam- 

ination..         ..          ..  356 

—  sugar  gelatin  media        . .  358 
Human  versus  bovine  tubercle 

bacilli  . .          . .         277,  390 

-  tubercle  bacilli     . .         277,  290 

..  293 

294,  392 


—  tuberculosis 

table  of  cases  of 

Humanized  condensed  milk 

—  cow's  milk 
Humus 

Hydrogen  peroxide  in  milk 


Ice,  analysis  of 
Ilosvay's  naphthylamine  test 
for  nitrites  in  water  . . 
Immune  body 
Immunity 

—  absolute    . . 

—  acquired    . .  . .         181, 

—  and  anaphylaxis 

—  artificial     . . 

—  natural 

—  specific 

—  theories  of 
Immunization,  active 

—  passive 

—  anthrax 


—  tetanus 

—  unit 
Incubator     test     for    sewage 

and  sewage  effluents 


85 
83 
7i 
82 

53 

48 
203 
181 
180 
183 
180 
184 
181 
183 
198 
184 
188 
307 
315 
191 

57 


400 


INDEX 


acids 


85 


111 


India  ink  method 

Indian  Plague  Commission 

Indicators,  classification  of 

—  neutral  point 

—  sensitiveness  of     . . 
Indigo  process  for  nitrates  in 

water 
Indol  formation,  tests  for 
Infant  foods,  analysis  of 

—  —  preparation    of 

table  of 

Infection 

—  in  children  and  adults 

—  conditioned  by 

—  effects  of  . . 

—  mode  of  action 
Infective  disease,   definition 
Influenza  bacillus 
Inhalation  of  plague 
Inoculation  of  animals 

—  plague 

Inosite  or  muscle  sugar 
Insoluble      fatty      acids 

butter-fat 

—  volatile     fatty 

butter-fat 

Internal  anthrax 

Interpretation    of 

water  analysis 

Intestinal  plague 

Intracellular  toxins 

Inulin 

Inunction  test  with  tubercu- 
lin          

Inversion  of  saccharose 

Invert  sugar  . . 

syrup 

Invertase 

Iodine  absorption  of  butter  fat 

—  test  for  formalin  . . 

sulphites 

Iron  in  water,  estimation  of 

amount 

—  —  presence     of     causing 

taste     . . 

tests  for 

Isolation     of     B.     anthracis 

from  hairs 

—  B.  coli  from  water 

—  sp.  cholerae  . .         323 

—  tetanus  bacillus 
Isolysins 
Itch,  dhobie's 


PAGE 
328 

259 
IO 
II 
IO 

52 
159 

86 
86 
87 
173 
295 
174 
174 
174 
173 
243 
257 
169 
257 
106 

91 


90 
304,  306 
results  of 

53 
260 
176 
105 

281 
103 
103 
no 

332 

94 
136 
121 

28 

29 
125 

307 
353 
1  324 
3ii 
211 
338 


PAGE 

Kefir              375 

Kjeldahl's   method   for    total 

nitrogen  in  sewage      . .  59 

organic  matter  in  water 

40,  46 

total  nitrogen  in  milk  78 

Klebs-Loeffler  bacillus          . .  246 

Koch's  bacillary  emulsion   . .  281 

—  postulates..           ..          ..  150 

—  steam  sterilizer     . .          . .  155 

—  tuberculin..          ..         279,  281 

Koch-Weeks  bacillus             . .  244 
Kola     ..          ..          ..          ..119 

Koppeschaar's  process          . .  137 

Koumiss           . .          . .          . .  375 


103 


Jenner's  stain  . .  . .      167 

Johne's  bacillus  . .  285,  372 

Jorissen's   test   for   formalde- 
hyde in  milk  . .  . .        81 


Lacmoid  papers,  use  of 
Lactic  acid  bacillus  . . 
Lactose  agar 

—  broth 

—  in  milk 

—  or  sugar  of  milk . . 
Lactose-nutrose-agar 
Laevulose         . . 
Lamirasacsal  streptococci 
Lanc^-acetone-baryta  method 
Lancet  Commission  on  Stand 

ardization     of     Disin 
fectants 

—  bacteriological  test 

—  reports  on  plague  in  China 
Lead  in' water,  determination 

of  amount 

presence  of 

Leffmann-Beam  process  for  fat 

in  milk 
Leishman's  stain 
Lemon  juice 
Leprosy  bacillus 
Letts  and  Blake  process 
Leucocidin 
Leucocyte  extract 
Leucocytosis 
Levaditi's  method 
Light  oils  in  carbolic  acid 
Lime  juice 

Lime  in  water,  tests   for 
Liquefaction  of  gelatin 
Litmus  lactose  agar  . . 

—  milk,  glycerin 

—  solution,  reactions  of 

—  whey 
Local     inspection     of    water 

supplies 
Lockjaw 
Loe filer's  blood  serum 

—  medium 

—  methylene-blue     . . 


12 

242 

153 

152 

77 

104,  109 

239 
104 

359 
139 


139 
380 
263 

27 
25 

75 
168 
119 
285 

58 
177 
196 
182 
328 
138 
119 
29,  30 
159 
153 
3907 

11 
154 

53 
3ii 
246 

239 
161 


INDEX 


401 


Low  fermentation 
"  Lumpy  jaw  " 
Lupus 
Lymphocytosis 


PAGE 

333 

286 
295 
182 


352 
153 


76 
..  286 
••  239 
..  124 
••  3i7 
304,  306 

•  •      252 

•  •      252 
..      125 

122 

228-9 

••      332 

104 

344 


56 
105 
105 
no 


MacConkey's  broth,  reaction 
of  certain  bacteria  with 

—  media 
Maceration  process  for  fat  in 

milk 
Madura  foot 
Malachite-green  medium 
Malic  acid  in  cider  vinegar  . . 
Malignant  oedema  bacillus 

—  pustule 
Mallein 

—  test 
Malt  beer 

—  vinegar 
Malta  fever 
Maltase 
Maltose 

—  media         . .  . .        341 
Manchester  Ship  Canal,  stand- 
ard for  effluents 

"  Manna  " 
Mannite 
Maple  syrup 
Magnesia  in  water,  tests  for  29,  30 
Maragliano's  serum   . .  . .      282 

Margarine  v.  butter   . .  . .        95 

Margarine  in  butter,  calcula- 
tion of  amount  of     . .        89 

—  fats,  Reichert- Wollny  figure 

of  89 

Marmorek's  serum     .  .  . .      282 

Marmot  . .  . .  . .      264 

Mate     . .  . .  . .  . .      119 

McCrorie's  flagella  stain       . .      165 
M'Fadyean's  test       . .  . .     308 

Measuring  of  solutions  . .        12 

Meat  extracts  and  essences..      112 
Media,  bacteriological,  classified  151 

—  gelatin  sugar  (Houston's)       358 
Medium,  Raulin's 

—  Sabouraud's 
Megalosporon 
Mel  depuratum  B.P. . . 
Melitose  or  raffinose . . 
Meningococcus 
Metaphenylene-diamine  solu 

tion 
Metchnikoff's  bacteriotherapy 

—  phagocytic  theory 

—  spirillum    . . 
Methods  of  anaerobic  culture 

—  cultural 
Methyl  alcohol 


336 
34i 
343 
in 
105 
226 


47 
374 
198 
324 
310 
155 
134 


PAGE 

Methyl-orange    solution,    re- 
actions of        ..          ..  11 
Methylene-blue,  carbol         . .  161 

—  Loefner's    . .          . .          . .  161 

—  reaction  (in  anthrax)  . .  308 
Metric  system             . .          . .  14 

—  —  factors  for  conversion  of  15 

imperial  equivalents  of  16 

Micro-organism  of  syphilis  . .  327 

Micrococci       . .          . .          . .  220 

Micrococcus  catarrhalis         ..  228 

—  lanceolatus            . .          . .  224 

—  melitensis  . .          . .          . .  228 

—  tetragenus  . .  . .  228 
Microscopic  characters  of  coffee 

and  chicory     ..          ..  114 
tea  leaves        ..          ..  117 

—  examination  of  milk  . .  79 
Microspironema  pallidum  . .  327 
Microsporon    . .          . .          . .  343 

—  audouini    .  .          . .          . .  343 

—  furfur         . .          . .          . .  337 

—  minutissimum  . .  . .  338 
Midden  towns  sewage,  analysis 

of          56 

Milk,  actinomyces  in             . .  370 

—  alcoholic  fermentation  of  375 

—  ash  of        . .          . .          . .  74 

—  B.  enteritidis  sporogenes  in  368 

—  bacteriological  examination  366 
standards         . .          . .  367 

—  benzoates  in          . .          . .  82 

—  blue            375 

—  boiled         . .          . .          . .  79 

—  borax  in     . .  80  and  errata 

—  buddeized              . .          . .  83 

—  butter         . .          . .          . .  375 

—  butyric  acid  fermentation 

of          374 

—  calculation  of  adulteration  76 

—  cane  sugar  in       . .          . .  78 

—  centuf legalization  of       . .  373 

—  condensed             . .          . .  85 

—  cream  in    . .          . .          . .  73 

—  dirt  in         . .          . .          . .  84 

—  diseases  of             . .          . .  375 

—  dried          . .          . .          . .  83 

—  enumeration  of  bacteria  in  367 

—  examination  of     . .          . .  73 

—  faecal  organisms  in           . .  368 

—  fat  in         . .  . .  74-76 

—  fermentations  of  . .          . .  373 

—  "  fore  "       . .          . .          . .  369 

—  glycerin  litmus     .  .          . .  390 

—  green          375 

—  homogenized         . .          . .  83 

—  humanized  . .  . .  83 
condensed        . .          . .  85 


402 


INDEX 


PAGE 

Milk,  Johne's  bacillus  in       . .  372 

—  lactic  acid  fermentation  of  373 

—  lactose  in  . .          . .          . .  77 

—  leucocyte  test       . .          . .  373 

—  media         . .          . .        154,  390 

—  microscopic  examination  of  79 

—  "  mid "      . .          . .          . .  369 

—  nitrogen  in            . .  78 

—  pasteurized            . .          . .  83 

—  physical  characters  of     . .  73 

—  preservatives  in     79  and  errata 

—  —  in,  new  regulations    . .  388 

—  proteids  in  . .  79 
■ —  reaction  of           • .          . .  73 

—  red              375 

—  ropy           375 

—  slimy          . .          . .          . .  375 

—  soapy         375 

—  solids  not  fat        . .          . .  76 

—  specific  gravity  of           . .  73 

—  streptococci  in     . .          . .  372 

—  "  strippings  "        . .          . .  369 

—  total  solids  of       . .          . .  73 

—  tubercle  bacilli  in  . .  368 
—  deposit       . .          . .  370 

—  from  tubercular  udders  . .  369 

—  yellow        375 

Millinormal    solution,    defini- 
tion of . .          . .          . .  8 

Mineral  acid,  tests  for  free  . .  121 

—  waters,  analysis  of  . .  53 
Mines,  table  of  gases  in  . .  70 
Minimum  lethal  dose  . .  190 
Minute  bacilli  . .  . .  243 
Modes  of  study  of  bacteria  . .  156 
Modification  of  tubercle  bacilli 

278,  298 

Moist  heat  sterilization         . .  155 

Moisture  in  bread     . .          . .  100 

Molasses           ..          ..          ..  no 

Monosaccharids  or  glucoses. .  102 

Morax-Axenfeld  diplobacillus  245 

Moro's  test     . .          . .          . .  281 

Mother's  milk,  average  com- 
position of                   . .  86 
Moulds             . .          . .          . .  334 

—  head           334 

—  knob           335 

—  pencil         . .          . .          . .  336 

—  ringworm  . .          . .          . .  341 

- —  and  yeasts             . .          . .  330 

Mucor  mucedo           . .          . .  334 

Muguet            . .          . .          . .  340 

Mulkowal  outbreak  (tetanus)  314 

Mus  decumanus          . .          . .  258 

—  rattus         . .          . .          . .  258 

Muscle  sugar  or  inosite         . .  106 

Mustard,  adulterations  of    . .  112 


Mustard,  composition  of      ..  112 

Mycelium        . .          . .          . .  334 

Mycetoma       . .          . .          . .  286 

Mycomycetes              . .          .  .  334 

Mycoses           . .          . .          . .  330 

"  Nail-head  "  growth          . .  241 

"  Naked  "  bacteria   . .          . .  380 

Naphthylamine  solution       .  .  48 

—  test  for  nitrites  in  water . .  47 
Natural  anaphylaxis             . .  218 

—  immunity  . .          . .          . .  181 

Negative  phase           . .          . .  195 

Nessler's    solution,     prepara- 
tion of . .          . .          . .  40 

Nesslenzing,  defined..          ..  27 
Neutral  oils  in  disinfectants 

140,  141,  142 

—  point  of  indicators  . .  n 
Neutrality  indicators  . .  10 
"  New  butter  value "  . .  91 
New  tuberculin  . .  . .  281 
Nitrates  in  water,  tests  for . .  49 
Nitrites  in  flour  (from  bleach- 
ing)         99 

—  and  nitrates  in  water     . .  47 

—  in  water,  tests  for           . .  47 
Nitrogen  in  meat  extracts  . .  112 

—  milk            78 

—  peroxide,  use  of,  in  bleach- 

ing flour           . .          . .  99 

Nitroso-indol  reaction          . .  159 

Nocard's  experiments           . .  278 

Non-standardized  solutions..  9 

Normal  diphtheria  antitoxin  191 

—  solutions    . .          . .          . .  7 

definition  of   . .          . .  8 

Noxious    emanations    in    air, 

detection  of    . .          . .  67 

Nursing  of  plague     . .          . .  263 

Nutrient  agar             . .          . .  153 

—  broth          . .          . .          . .  151 

—  gelatin        . .          . .          . .  152 

Oidium  albicans        . .          . .  340 

Oil  of  theobroma       ..          ..  117 

Old  tuberculin           . .          . .  279 

Ophthalmo-tuberculin  reaction  281 

Opsonic  action           . .          . .  193 

—  co-efficient  of  extinction  194 

—  estimation,     Leishman's 

method             . .          . .  193 
Wright's  method       . .  194 

—  index          . .          . .          . .  194 

Opsonins         . .          . .         183,  193 

Organic  carbon  by  Frankland's 

method            . .          . .  38 


INDEX 


403 


l' AGE 

Organic,  matter  in  air  -  . .  66 
water,  estimation  of..  38 

—  — by  Forschammer 

process  39,  44 

—  —  —  —  Frankland's 

method        . .  38 

Kjeldahl's  process 

40,  46 

—  —  —  —  Wanklyn's 

method  39,  40 

—  nitrogen   by    Frankland's 

method  . .  . .  38 
ratio     to     albuminoid 

ammonia          . .          . .  55 

Original  gravity  of  beer  wort  128 

—  tuberculin  . .  . .  279 
Oxalic  acid,  standard  solution 

of,  for  baryta  32,  63 

carbonates       . .  31 

Oxidizable  matter  in  air       . .  66 

Oxygen  in  air             . .          . .  69 

—  absorption     by     organic 

matter  in  water          . .  44 

—  (dissolved)  in  water  . .  33 
Ozaena  bacillus  . .  . .  241 
Ozone  in  air  . .          . .          . .  66 

Pappenheim's  stain  . .          . .  285 

Paracolon  bacilli        . .          . .  236 

Paraffin-section  staining      . .  166 

—  wax,  melting-point  of     ..  in 
Paraguay  tea..          ..          ..  119 

Parasites  in  cereals  . .          . .  98 

Paratyphoid  "  A  "     . .          . .  236 

—  "B"           236 

—  bacilli         . .          . .          . .  236 

Park  &  Krumwiede's  research 

on  tubercle  bacilli — 

Tables  of  results           278,  392 

Conclusions         . .          . .  390 

Cultural  characters        . .  391 

Passive  immunization           . .  188 

Pasteurized  milk        . .          . .  83 

Pathogenic   penicillia            . .  337 

—  yeasts        333 

Pathology  of  plague  in  China  268 
Pavy-Fehling  method  for  sugar 

estimation       . .          . .  97 

Peas,  tests  for  copper  in       . .  112 

Peaty  colour  and  test  for  lead  28 

Penicillium      . .          . .          . .  336 

—  glaucum     . .          . .          . .  336 

Pepper,  adulteration  of       . .  112 

—  composition  of  ..  ..  112 
Peptone  water  ..  ..  153 
Percentage  index  . .  . .  194 
Permanganate  of  potash  . .  136 
Perry    . .          . .          . .          . .  132 


Petri's  method   of  air  exam- 

tion 
Petruschky's  litmus  whey 
Pettenkofer's      method      for 

estimation  of  C02  in  air 
Pfeiffer's  phenomenon 

—  reaction 
Phagocytic  index 
Phagocytosis 
Phenol,    bromine    absorption 

process  . .         141, 

—  tests  f or     . . 
Phenol-sulphonic  acid,  prepara- 
tion of  . .  10,  50 

—  test  for  nitrates  in  water       50 
Phenolic  bodies  in  disinfectants 

140,   142 
Phenoloids,     relation    of,     to 
carbolic  acid  co-efficient 
Phenolphthalein  solution,  re- 
actions of 
Phenomenon  of  Arthus 

—  Pfeiffer 

—  Theobald  Smith  . . 
Phloroglucinol  test  for  form- 
aldehyde in  milk 

Phosphates  in  water,  tests  for 

29, 
Phosphoric  acid  in  cereals  . 
Phycomycetes 
Physical  characters  of  milk. 
Pinot's    method    for    CI    in 

bleaching  powder 
Piquette 

Pityriasis  versicolor 
Plague  bacillus 

—  in  California 

—  China 
summary  of  conclusions 

of      .. 

symptoms  of 

Egypt 


—  Glasgow     . . 

—  Harbin 

—  India 

—  Manchuria 

—  in  marmots 

—  in  rats 

—  spots 

—  in  squirrels 

—  Suffolk 
Pneumobacillus 
Pneumococcus 
Pneumonic  plague 

in  China 

Poisonous   metals 

cheese  . . 

—  —  vinegar 


271 
265 
259 
254 
266 
260 
263 
264 

258,  262,  269 
260 
255 
254 
240 
224 
259 
263 


rind  of 


26A 


363 
154 

63 
191 
322 
194 
181 

i37 
i37 


i43 

11 
214 
191 
214 


30 

97 

334 

73 

135 
131 
337 
254 
255 
263 


96 

124 


404 


INDEX 


PAGE 

Poisonous  metals  in  water   . .  25 

qualitative  tests  . .  25 

quantitative  tests  26 

Poisons,  animal  and  vegetable  178 

— ■  bacterial    . .          . .          . .  175 

Poivrette,  in  pepper..          ..  112 

Polarimeter  test  for  sugar   . .  107 

Polariscope  test  for  butter  . .  94 

Polenske  figure  for  fats        . .  91 

—  process  for  butter-fat  . .  90 
Polychrome  stains  . .  . .  167 
Polymorphism  . .  . .  149 
Polysaccharids  or  starches  . .  102 
Porges- Meier  reaction  . .  210 
Portals  of  entry   of  tubercle 

bacilli  . .          . .          . .  279 

Positive  phase            . .          . .  196 

Post  mortem  of  rat  (plague)  262 

Postulates,  Koch's     . .          . .  150 

Potassium  permanganate,alka- 

line  solution  of          . .  43 

—  —  normal  solution  of  . .  9 
standard    solution    for 

oxygen  absorption     . .  45 

Potato  bacillus           . .          . .  310 

—  medium     . .          . .          . .  154 

Precipitation  . .          . .          . .  192 

Precipitins       . .          . .          . .  192 

Preservatives  in  beer            . .  129 

—  cream  . .  84  and  errata 
new  regulations          . .  388 

—  milk  . .  79  and  errata 

new  regulations         . .  388 

Presumptive  B.  coli  test      . .  352 

Prevention  of  anthrax  . .  309 
Primary  abdominal  tuberculosis  293 

—  —  —  table  of  cases  of  . .  294 
Products  of  bacterial  activity  172 
Proof  spirit     . .          . .          . .  133 

Prophylaxis  of  plague          . .  262 

—  tetanus       . .          . .          . .  315 

Proteids  in  milk         . .          . .  79 

Proteinochrome  formation  by 

bacteria           . .          . .  159 

Protozoa          . .          . .          . .  171 

Ptomaines       . .          . .          . .  172 

Public  Health,  regulations  for 

Diploma  in,     . .          . .  384 

Pulex  cheopis             . .          . .  257 

—  irritans       . .          . .          . .  259 

Pus,  examination  of . .          . .  224 

Putrescin         . .          . .          . .  173 

Qualitative  chemical  analysis  5 

Quantitative  chemical  analysis  5 

Quarantine  in  plague  in  China  270 

Quarter  evil    . .          . .          . .  318 

Quinine  injections  and  tetanus  314 


Raffinose  or  melitose         . .  105 

Rainfall  and  bacteria  content  351 

Rat  agar         . .          . .          . .  255 

—  buboes       . .          . .          . .  262 

—  flea             . .          . .          . .  257 

Rats  in  China            . .          . .  269 

Raulin's  liquid  medium        . .  336 

Rauschbrand  . .         . .          . .  318 

Raw     River    Thames  water, 

bacteria  in      . .         350,  351 

Reaction  of  milk       . .          . .  73 

Reaction,  Smith         . .          . .  390 

Reactions  of  sewage  bacteria, 

table  of            ..         354,  355 

Rebipelagar     . .          . .          . .  350 

Recommendations   of  Tuber- 
culosis Commission       302-3 

Reference  terms,  reply  to     . .  299 

Refractive  index  of  butter-fat  92 
Regulations    for    Diploma   in 

Public  Health            ..  384 
Reichert-Wollny    figures    for 
butter   and  margarine 

fats 89 

coco-nut  fat    . .          . .  89 

fish-oils            . .          . .  90 

—  —  palm  oil           . .          . .  90 

—  process  for  butter-fat       . .  88 
Reinsch's  test  for  arsenic  in 

beer      . .          . .          . .  129 

Relapsing  fever           . .          . .  326 

Rhinoscleroma  bacillus         .  .  242 
Richmond's  test  for  boric  acid 

in  milk     . .      errata  and  80 

Rideal-Walker  test    . .          . .  379 

Ringworm  fungi         . .          . .  341 

table  of            . .          . .  342 

Romanowsky  stain   . .          . .  167 

Rosolic  acid  solution,  reactions 

of           ..           ..           ..  12 

Rotatory  power,  specific     . .  107 
Royal  Commission  on  Tuber- 
culosis             . .          . .  288 

Rum     . .          . .          . .          . .  132 

Saccharate  of  lime  in  cream  84 

Saccharates  or  sucrates        . .  103 

Saccharin  in  beer       . .          .  .  129 

Saccharomyces  albicans        . .  340 

—  cerevisiae    . .          . .          . .  322 

—  niger           333 

—  rosaceus     . .          . .          . .  333 

Saccharimeter  test  for  sugars  107 

Saccharose       . .          . .          . .  103 

—  in  cane  sugar       . .          . .  107 

—  gravimetric  estimation  of  108 

—  volumetric  estimation  of  108 
Sagin  B.  coli  . .          . .          . .  357 


INDEX 


405 


Salicin 

Salicylic  acid  in  beer 

milk 

test  for 

Sabouraud's  medium 
Salt  agar 

—  in  butter 

Salts  with  enlarged  molecules 
Sampling  of  air  for  analysis 

—  sewage  and  sewage  effluent 

for  analysis     . . 

—  water  for  analysis  21, 
Saponification    equivalent    of 

butter  fat 
definition  of   . . 

—  value,  definition  of 

—  values     and    equivalents, 

table  of 
Schiff's  test  for  formaldehyde 

in  milk 
Schizomycetes 
Sclavo's  serum 
Search  for  B.  coli  in  water  . . 

—  —  tetani  (wounds) 
Sedgwick  and  Tucker's  method 

of  air  examination     . . 
Seminormal  solution,  definition 

of  

Sensitiveness  of  indicators  . . 
Separated  milks 
Septicemic  plague 
Sera,  antitoxic 

—  antituberculous 
Serum,  anticholera     . . 

—  antidiphtheritic    . . 

—  antiplague 

—  antitetanus 

—  blood 

—  diagnosis  in  plague 

—  disease 

—  Margliano's 

—  Marmorek's 

—  precipitation 

—  Sclavo's     . . 

—  sickness 

—  Sobernheim's 

—  Yersin's ... 
Sesame  oil  in  butter  fat 
Sewage  Commission,  standards 

for  effluents    . . 

—  effluents,     bacteriological 

examination  of 

—  percentage  purification  of, 

see  tables  . .         56,  61 

—  and    sewage    effluents, 

Birmingham  sewage..        56 

—  —  —  examination  of     . .        57 

—  —  —  Glasgow   analyses         61 


PAGE 
I06 
129 
82 


121 

341 

255 

88 


Sewage  and  sewage  effluents, 
incubator  test 

Kjeldahl's    method 

for  total  nitrogen 

Letts     and     Blake 

process 
midden  towns  sewage 
—  Staffordshire  experi- 
ments 

standards  in 

suspended  solids  in 

—  —  —  table  of  analyses . . 
watercloset     towns 

sewage 
Shake  culture 

Shellfish  

Shiga- Kruse  group 

Shiga's  bacillus 

Smegma  bacillus 

Smith  reaction 

Soap  solution,  preparation  and 

standardization  of  35,  36 
Sodium  sulphite  . .  121,  139 
Soil,  bacteriological  examina 

tion  of . . 

—  organisms  found  in 

—  temperature 
Soils,  examination  of 
Solids-not-fat,  in  milk 
Soluble  cocoa. . 

—  toxins 

Solution,    standard   arsenious 
Solutions,    normal     . . 

—  standard 
Soor 
Sorbite 
Sorghum 

—  juice 
Soxhlet    apparatus,    used   in 

Adams'  process  . .  75 
Specific  gravity  of  butter-fat  92 
milk      . .          . .  . .        73 

—  immunity  . .  . .  . .      183 

to  bacteria      . .  . .      185 

toxins  . .  . .  . .      185 

—  rotatory  power     . .  . .      107 

of  dextrose  ..      107 

glucose  syrup       . .      no 

—  —  —  golden  syrup         . .      no 

honey  ..  ..      in 

— laevulose     . .  . .      107 

365       —  —  —  saccharose..  ..      107 

Specimen  analyses  of  waters         55 
Spirilloses        . .  . .  . .      326 

Spirillum  aquatilis   (Gunther)     325 

—  cholera?  asiaticas  . .  . .      321 

—  danubicus      . .    . .   325 

—  deneke   . .    . .    . .  325 


57 
345 

93 
93 
93 

93 

81 
171 
307 
35i 
316 

364 


10 

85 
260 
189 
282 
323 
248 
261 
315 
154 
261 
212 
282 
282 
192 
307 
212 
307 
261 

94 
56 


57 

59 

58 
56 

56 
56 
57 
56 

56 
157 
376 
238 
237 
285 
390 


365 

365 

72 

7i 

76 

117 

176 

135 

7 

6 

340 

106 

103 

no 


406 


INDEX 


Spirillum  of  Finkler  and  Prior     325 

Starch  iodine  test  for  nitrites 

—  Massowah . . 

•      325 

in  water           . . 

47 

—  Metchnikovi 

•      324 

—  paste  in  cream 

84 

—  Obermeieri 

326 

in  honey 

in 

—  phosphorescent     .  . 

•      325 

—  sugar  or  glucose      103,  104 

109 

—  of  relapsing  fever 

.      326 

—  sugaring  of  by  diastase  . . 

105 

—  tyrogenum 

•      325 

Starches,  beers  from . . 

127 

Spirit     indication,     table     of 

—  examination  of 

IOI 

acid  values 

•      145 

—  morphology  of 

IOI 

—  —  —  values  of  . . 

146 

—  or  polysaccharids 

102 

—  proof 

•      133 

Steam  in  sterilization 

155 

Spirits.. 

•      132 

Sterilization 

155 

Spirochaeta  pallida     . . 

•      327 

Streak  culture 

157 

—  pertenuis   . . 

•      329 

Streptococci    . . 

222 

—  recurrentis 

.      326 

—  lamirasacsal 

359 

—  refringens  . . 

•      327 

—  in  water 

358 

—  Vincenti 

•      327 

Streptococcus  lacticus  (Kruse) 

Spirochetes 

•      326 

242, 

374 

Spirochetoses 

326 

—  mucosus 

226 

Spontaneous  generation 

148 

Subsoil 

7i 

Sporangium 

334 

—  water 

72 

Spore  staining 

164 

Substance  sensibilatrice 

203 

Sporing  bacilli 

304 

Sucrose 

103 

Sporotrichosis 

338 

Sugar  barley  . . 

103 

Sporotrichum  Beurmanni     . 

338 

—  beet 

103 

Spread  of  plague  (reference) 

263 

—  cane            . .          . .         103, 

106 

—  —  in  China 

266 

—  Demerara 

109 

Stab  culture 

157 

—  fruit            

104 

Stability  in  culture  of  bovine 

—  grape          . .            103,  104, 

109 

tubercle  bacilli 

290 

—  maple 

103 

human  tubercle  bacilli     291 

—  milk            . .          . .         104, 

109 

Staffordshire   experiments   in 

—  mite,  or  Acarus  sacchari 

109 

sewage  treatment 

56 

—  muscle 

106 

Staining  reactions  and  methods  161 

—  palm 

103 

—  by  Romanowsky  stains  . 

168 

—  starch 

109 

"  Stalactite  "  growth 

256 

Sugars  or  disaccharids 

102 

Standard  arsenious  solution 

135 

Sugaring  of  starch  by  diastase 

105 

—  ferrous  sulphate  . . 

137 

Sulphanilic  acid  solution     . . 

48 

—  nitrite  solution  for  water 

Sulphates  in  water,  tests  for 

analysis 

47 

2C 

■   30 

—  soap  solution,  preparation 

Sulphites  in  milk 

82 

and  standardization 

35,   36 

—  tests  f or     . . 

121 

—  solution      of      ammonium 

Sulphuretted     hydrogen     in 

chloride 

41 

wines   . . 

7o 

oxalic  acid  for  baryta 

32,  63 

water 

34 

carbonates 

3i 

Sulphuric  acid  in  vinegar    124, 

125 

—  —  potassium  permangan 

Sulphurous  acid        ...        121 

139 

ate  for  oxygen  absorp 

Surface  soil 

7i 

tion 

45 

Suspended  matter  in  air 

69 

—  solutions 

6 

—  solids  in  sewage  effluents 

57 

Standards  in  sewage  effluents 

56 

Sweet  wine 

130 

Standardization  of  disinfect 

Syrup,  golden 

no 

ants,    Lancet    Commis 

—  invert  sugar 

no 

sion 

139 

—  maple 

no 

—  sera 

189 

Staphylococci 

220 

Tabarie's  method  for  alcohol 

Starch  or  amylum 

104 

in  beer 

128 

—  in  butter    . . 

94 

Table  of  anaerobes  (sporing) 

320 

INDEX 


407 


PAGE 

Table  of  animal  temperatures     170 

—  carbohydrates    and   allied 

substances       . .  . .      106 

—  characteristics    of    colon- 

typhoid  group            . .  240 

—  composition  of  infant  foods  87 

—  degrees  of  spirit  indication  146 

—  gelatin-liquefying     organ- 

isms     . .  . .  . .      160 

—  Glaisher's  factors  . .      144 

—  gram-positive    and    gram- 

negative  organisms    . .      162 

—  to    illustrate    Frankland's 

method,  applied  to  water    39 

—  of      principal      ringworm 

fungi 342 

—  reactions  of  sewage  bacteria 

354,  355 

—  researches    by    Park    and 

Krumwiede      . .         278,  392 

—  saponification  equivalents       93 

—  sewage  and  sewage  efflu- 
ent analyses   . .  56,  61 


—  short  alcohol 

—  of  spirit   indication  value 

of  acid  in  beer 

—  water  analyses     . . 

—  weights  of  1  eft.  of  water 

vapour 
Tannin  in  tea 
Tarabagan 

—  disease 
Tartaric  acid,  test  for 

—  —  in  wine 
Tatten-Thomson     test      for 

formaldehyde  in  milk 
Tea 

—  fresh  v.   exhausted  leaves 
Temperature  in  soil 
Temporary  hardness  in  water 
Terms  of  reference,  replies  to 
Tetanolysin 
Tetanospasmin 
Tetanus 

—  bacillus 

—  toxin 

Theobromine  in  cocoa 
Theories  of  immunity 
Thrush 
Tidy's    process    for    organic 


144 

145 

55 

145 
116 
264 
264 
122 
131 

81 
115 
117 
72 
37 
299 
314 
314 
3ii 
3ii 
314 
118 
198 
340 


matter  in  water 
Tinned  peas 
Titration,  definition  of 
Top  yeast 
Torula  niger   . . 
—  rosea 
Torulae 
Total  hardness  in  water 


39 


127 


44 
112 
6 
333 
333 
333 
333 
37,  38 


Total  secondary  products    . . 

—  solids  of  milk 
Toxins . . 

—  extracellular,      true,      or 

soluble . . 

—  intracellular  or  endo- 

—  nature  of  . . 
Transmission  of  plague 
Treponema  pallidum 

—  pertenue 
Trichophyton  mentagrophytes 

—  Sabouraudi 

—  tonsurans 

Trommsdorff's  leucocyte  test 
True  toxins 
Tubercle  bacilli 

—  —  avian  type      . .         278, 
bovine  type      277,  289,  390 

—  —  human  277,  290,  390 
.  (Park  and  Krumwiede's 

researches)  277,  390 

—  —  in  fish  . . 

—  —  in  milk 
pus,  faeces  and  tissues 

—  —  sputum 

urine     . . 

Tuberculin,  new 

—  -O   . . 

—  original 

—  -R  .. 

—  tests  in  cattle 

—  vaccine  therapy 
Tuberculosis  in  birds 

—  Commission,  recommenda 

tions     . .  . .         302 

—  in  home  cattle 

—  horses 

—  sheep 

—  swine 

—  various  animals 
Turmeric  solution,  reactions  of 
Typhoid  bacillus 
Typical  B.  coli  (British  Com 

mittee)  . .  . .     349 
(Houston) . .           . .     356 

—  —  —  communis  . .     356 

Unicellular     micro-organ- 
isms     . .          . .          . .  170 

Vaccination  for  anthrax       . .  307 

Vaccine,  anticholera  . .          . .  323 

—  antigonococcal      . .          . .  228 

—  antiplague             • .         261,  270 

—  antistaphylococcal            . .  221 

—  antitubercle          . .          . .  282 

—  therapy      . .          . .          . .  194 

for  typhoid  fever       . .  233 

Valenta  test  for  butter-fat  . .  92 


134 

73 

175 

176 

176 
178 
257 
327 
329 
343 
343 
342 
373 
176 
273 
291 


279 
284 
284 
283 
284 
281 
281 

279 
281 
283 
282 
296 

303 

276 
296 
276 
295 
296 
12 
231 


408 


INDEX 


PAGE 

PAGE 

Vibrio  choleras 

321 

Water  analysis,  nitrates       . .       49 

Vincent's 

angina 

247, 

327 

nitrites             . .          . .       47 

Vinegar 

122 

and  nitrates          . .       47 

—  cider 

123 

volatile  solids             . .       23 

—  malt 

122 

—  —  organic  matter,  various 

—  white 

123 

methods  of  estimation       38 

—  wine 

122 

oxygen  absorption     . .       44 

—  wood 

123 

—  —  permanent     or     fixed 

Viscogen 

in  cream     . . 

84 

hardness          . .             37,  38 

Vitality   in   culture   of   avian 

tubercle  bacilli           . .  293 

Voges-Proskauer  reaction    238,  355 
Volatile  soluble  fatty  acids  in 

butter  fat         . .          ...  88 

Volume  and  density  of  gases  14 

Volumetric  analysis  . .          . .  5 

—  —  requirements  for        . .  6 

—  methods     . .          . .          . .  6 

Von  Pirquet's  test      . .          . .  280 

Wanklyn's    method    for    or- 
ganic matter  in  water 

39,  40 

Wassermann  reaction           . .  328 

—  test             . .          . .        205,  209 

—  —  value  of           . .          . .  209 
Water  analysis           . .          . .  19 

albuminoid  ammonia  43 

arsenic,  presence  of  . .  26 

bacteriological     exam- 
ination            . .           21,  345 

bicarbonates   and  car- 
bonates           . .          . .  31 

chemical  examination 

20,  22 

chlorine            . .          . .  24 

collection  of  sample    21,  345 

—  —  dissolved  oxygen       . .  33 

solids          . .          . .  22 

estimation   of   amount 

of  copper  in   . .          . .  28 

■ of  iron  in        . .  28 

lead  in  27 

zinc  in  29 

fixed  solids      . .          . .  23 

Forschammer   process, 

for  organic  matter  39,  44 
Frankland's    method, 

for  organic  matter     . .  38 

—  —  free  carbon  dioxide   . .  30 

and  bicarbonate  32 

—  and  saline  ammonia  40 

hardness,  definition  of  34 

determination  of..  35 

interpretation  of  results  53 

Kjeldahl's  process,  for 

organic  matter  40,  46 
lime  and  magnesia      29,  30 


—  —  phosphates      . .  29,  30 

physical  examination  20,  22 

poisonous  metals       . .       25 

—  —  presence  of  lead,  copper, 

iron  or  zinc  . .  . .  25 
qualitative     tests     for 

poisonous  metals  . .  25 
quantitative    tests    for 

poisonous  metals  . .  26 
reaction  . .  . .       22 

—  —  specimen  analyses     . .       55 

sulphates         . .  29,  30 

sulphuretted  hydrogen       34 

temporary  and  remov- 
able hardness..  ..       37 

—  —  tin,  presence  of  . .       26 

total   hardness  37,  38 

solids  . .  . .       22 

Wanklyn's  method  for 

organic  matter  39,  40 
zinc,  presence  of        . .       26 

—  bacteria  found  in. .  ..      345 

—  bacteriological     examina- 

tion of  . .  . .     345 

methods  of  examination   346 

sampling  . .  . .     345 

standards         . .  . .     346 

—  in  butter 88 

—  of  crystallization . .  . .  8 

—  dilutions    . .  . .  . .      346 

—  enumeration  of  bacteria  in     349 

—  ground       . .  • .  . .       72 

—  isolation  of  B.  enteritidis 

sporogenes  . .  . .  360 
typhosus    . .          . .     360 

—  —  sp.  choleras      .  .  . .      324 

—  subsoil        . .  . .  . .        72 

—  tables  of  bacteria  in  350,  362 
Watercloset     towns     sewage, 

analysis  of  . .  . .        56 

Watercress      . .  . .  . .     376 

Weigert's  staining  methods 

166,  167 
Weighing  with   the   chemical 

balance  . .  . .       12 

—  and  measuring,  rules  as  to  12 
Weights,  atomic,  table  of  . .  15 
Werner -Schmidt    process    for 

fat  in  milk      . .  . .       74 


INDEX 


409 


Wheat  flour,  analysis  of 

Whey,  litmus 

Whisky 

White  damp  in  mines 

—  vinegar 

"  Whitening  "  of  sugar 
Whooping-cough  bacillus 
Widal's    reaction :     interpre 

tion  of  results 

in  typhoid  fever 

Wild  yeasts    . . 

Wine    . . 

— "  substitute . . 

—  vinegar 
Wood  spirit    . . 

—  vinegar 

Wool-sorter's  disease  304, 

Wort,  beer     . .  . .         126, 

agar     . . 

Wright's  vaccine  therapy   194, 


I'AGF. 

PAGE 

98 

Xerosis  bacillus 

247 

154 

132 

70 

123 

Yams 

Yeast  in  beer  . .           127,  332 

329 
333 

Yeasts.. 

33i 

IO9 

—  cultivated. . 

333 

244 

—  and  moulds 

330 

235 
234 

333 

—  pathogenic 

—  wild 

Yersin's  anti-plague  serum  . . 

333 

333 
261 

130 

i*3i 

Zinc  chloride . . 

139 

122 

—  in    water,    estimation    of 

134 

amount 

29 

123 

tests  for 

25 

306 

Zur  Nedden's  bacillus 

245 

128 

Zygospore 

335 

344 

Zymase 

332 

282 

9 


rM^    * 


